Molecular mechanism of menthol-induced TRPV5 channel inhibition

Domain: biology
Published: 2026-03-18
Editorial outcome: major_revision

Abstract
TRPV5 channels play a critical role in calcium homeostasis and are implicated in various pathophysiological
conditions. Here, we demonstrate that the monoterpene menthol, commonly used for pain and inflammation
management, is an inhibitor of TRPV5. Electrophysiology experiments reveal that menthol blocks ion
conduction through a slow blocker mechanism. Using single-particle cryo-EM, we determine the structure of
menthol-bound TRPV5, which shows menthol interacting with W583, a residue previously implicated in
channel permeation, gating and binding of endogenous modulators. These findings expand the repertoire of
TRPV5 modulators and suggest that menthol could serve as a scaffold for developing channel-targeting
modulators.

Introduction
TRP channels are important regulators of cation transport in a myriad of organisms and tissues. Vanilloid
TRP channels consist of subtypes TRPV1-6 and constitute one of the most diverse subfamilies, including
members activated by temperature, lipids, and natural compounds that participate in various important
physiological and pathophysiological processes. Most TRPV channels exert their actions through calcium
(Ca2+) signaling or the regulation of Ca2+ homeostasis1.
Among the TRPV channels, TRPV5 has one of the highest permeabilities for Ca2+ ions over monovalent
cations, with a relative Ca2+ to Na+ permeability ~1002, a property it shares with its homolog protein, TRPV6,
with 70% amino acid sequence homology between both proteins 3,4. These channels are mainly found in
Ca2+-absorbing epithelia, such as kidney and intestinal tissues, where their main function is the reabsorption
of Ca2+. TRPV5 is primarily expressed in kidney epithelial cells, specifically localized to the apical
membrane of cells of the distal convoluted tubules (DCT) and connecting tubules (CNT) of the kidney,
where it functions as a highly selective epithelial calcium channel, playing a crucial role in Ca2+ reabsorption.
While TRPV5 is predominantly found in the kidney, it is also expressed at lower levels in other tissues such
as brain and bone, where it has been shown to be essential for bone resorption by osteoclasts5,6.
TRPV5 channels, similarly to most members of the TRPV subfamily, form a functional tetramer at the cell
surface assembled by subunits containing six transmembrane domains with large intracellular N- and C-
termini. The pore domain consists of the S5, loop, and S6 segments of each subunit, which assemble
symmetrically in a four-fold structure to create a central ion conduction pathway7,8. The re-entrant pore loop
of each subunit forms the extracellular part of the pore and functions as a selectivity filter, determining ion
permeability, with a single aspartic residue at position 542 acting as a Ca2+ selectivity filter9,10. The S6
helices line the intracellular pore and form the gate at its narrowest part. TRPV5 inactivates in response to
Ca2+ entry, a process mediated by calmodulin (CaM) binding to the channel11 promoted by the interaction
between the last residue at the end of the S6 gate of the channel, the highly conserved W583, with a C-
terminal lysine of the CaM, which blocks the flow of ions through the pore7,12. The disruption of CaM binding
leads to increased TRPV5 expression at the plasma membrane by avoiding channel retention in the
endoplasmic reticulum13. The tryptophan residue W583 is highly conserved in TRPV5 across various
species, but it is absent in other members of the TRPV family and is crucial for channel gating14.
TRPV5 channels display marked inward rectification and are constitutively open at negative potentials,
allowing for passive entry of cations into cells at the resting membrane potential. Regulation of TRPV5 is
mainly carried out by the control of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipid concentration,
which binds between the N-linker, S4-S5 linker , and the S6 helix to keep the channel open12. Signaling by
vitamin D, which mostly regulates expression of protein at the plasma membrane15 and by parathyroid
hormone, which acts through PKA phosphorylation16, also regulate the channel. The long-chain lipid acyl-
coenzyme A (LC-CoA) is a highly efficient activator of TRPV5 that binds to the same site as the endogenous
ligand PI(4,5)P2 and induces very similar conformation changes to open the channel17.
TRPV5 channels have a relatively limited pharmacology, with only a few known additional endogenous
activators, such as PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) and the b-glucuronidase, Klotho, that
hydrolyzes extracellular sugar residues of TRPV518,19. Conversely, TRPV5 is inhibited by synthetic
compounds, including TH-117720, cannabinoids21, and a higher specificity inhibitor, ZINC1798899022.
Econazole and miconazole are antifungal agents that act as inhibitors of TRPV5. These compounds bind to
a specific pocket situated between the S4 and S5 transmembrane domains23, which is shared with TRPV6
channels24 and likely act as allosteric inhibitors of channel opening. Additionally, ruthenium red is a non-
selective inhibitor of many ion channels. In the case of TRPV5, it binds to the pore domain, likely blocking
conduction and stabilizing the channel in a closed state24,25.
Menthol is a highly-membrane permeable monoterpene alcohol derived from the mint plant (Mentha sp.)
and a known activator of cold-sensing TRPM8 channels26. Menthol is an interesting compound that has
been associated with renal toxicity and impaired intestinal mobility27,28, which could be mediated in part by
TRPV5 and TRPV6 ion channels. Menthol also activates or inhibits other TRP channels, such as TRPV3,
TRPV1 and TRPA1, although the structural mechanisms involved are not known29–32. TRPV5 and TRPV6
are only 30 % similar to other TRPVs which suggests that different mechanisms of modulation by menthol.
Given its potential effects on renal and gut physiology, and lack of mechanistic information, we explored
menthol’s actions on TRPV5 channels. Here we show that it acts as a blocker of TRPV5. We show that
menthol inhibits current through TRPV5 expressed in HEK293 cells by producing long-lived closures, likely
corresponding to slow blocking events. We solve the structure of rabbit TRPV5 in complex with menthol by
cryo-EM and find that a key determinant of menthol inhibition is a tryptophane residue at position 583
located at the intracellular entrance of the pore. Mutations W583Q and I575S readily reduce the effects of
menthol, suggesting that they are important mediators of the menthol interaction. Our results expand the
repertoire of inhibitors of TRPV5 channels and have implications to understand possible effects of menthol
in renal physiology.

Results
Menthol inhibits TRPV5 via a slow blocker mechanism.
First, we assessed the effect of menthol on the rabbit TRPV5 (rbTRPV5) by applying 500 μM menthol to the
external face of the membrane in outside-out patch configuration. Menthol rapidly inhibited more than 50%
of the current at all voltages without altering current kinetics (Figure 1a). This inhibitory effect was largely
reversible and occurred in a dose-dependent manner across all tested voltages (Figure 1b, c, d). Fitting the
dose-response curve with a Hill equation (Equation (1)), we determined a half-maximal inhibitory
concentration (IC ) of 142.6 μM and a Hill coefficient (n ) of 0.75, suggesting that a single menthol
50 H
molecule is sufficient to induce inhibition (Figure 1e).
Interestingly, menthol block of TRPV5 is slightly voltage dependent, with the blocked fraction reduced at
less negative potentials (Figure 1f). Because menthol does not carry a net charge, we sought to explain this
voltage-dependence by exploring the predictions of a multiple state model for block. We adapted a
previously published three state gating model of TRPV5 14 and incorporated open state block (Scheme I,
Figure 1g). To constrain the model, we measured the voltage-dependence of single-channel channel
openings, which indeed show that TRPV5 gating is intrinsically voltage dependent (Supplementary figure 1a
and c). Scheme I explains the voltage dependence of menthol block because it is coupled to the open state
and opening is itself voltage-dependent (Figure 1g). This model also accounts for both the voltage and
dose-dependence of block with the same set of parameters (Figure 1e).
Since the sequence of the inner pore region is highly conserved between mammalian TRPV5 and 6
channels, we examined the effect of menthol on the human TRPV5 orthologue and the related human
TRPV6 channel, finding that both channels were also blocked by menthol with similar efficacy
(Supplementary figure 2).
To further investigate the mechanism by which menthol inhibits TRPV5 currents, we analyzed single-
channel recordings. Recordings from patches containing a single TRPV5 channel at -100 mV showed that
gating was characterized by openings that occur in bursts (Figure 2a). The single-channel current amplitude
remained unaffected by menthol, as indicated by all-points amplitude histograms of selected current
segments (Figure 2b). In contrast, menthol increased both the frequency and duration of non-conducting
events in a dose-dependent manner. To quantify this effect, we idealized channel openings and compiled
open and closed dwell time histograms (Figure 2c and d). The gating behavior of TRPV5 shows three
exponential components in the closed-time distribution and two components in the open-time distribution
(Table 1 and 2). The number of exponential components in these distributions represents the minimum
number of closed or open states. In this case, data indicates there are three closed or non-conductive states
and one or two open states. These kinetics are consistent with the model in Scheme I (Figure 1g), although
this is not a definitive model.
Further analysis of single-channel recordings shows that menthol induces a concentration-dependent
lengthening of the longer closed-state durations, while reducing the slow component of the open-state
durations, consistent with menthol blocking ion conduction and shortening the length of bursts of openings
(Figure 2e, f and Table 1 and 2). These findings indicate that menthol inhibition occurs via a blocking
mechanism that does not alter single-channel current amplitude, a characteristic feature of slow channel
blockers33.
Structural mechanism of TRPV5 blockade by menthol.
To gain structural insight into the molecular mechanism by which menthol blocks TRPV5, we determined the
cryoEM structure of full-length rabbit TRPV5 in complex with menthol. A priori, it is unclear whether menthol
solely blocks the channel or also induces an alternative conformational state to close the channel. Given
that TRPV5 adopts an open state in the presence of PI(4,5)P , we initiated our experiments with a PIP₂-
2
activated channel and subsequently applied menthol to assess its effect, which are similar conditions to
those in which current recordings are performed. First, purified TRPV5 embedded in lipid nanodiscs was
incubated with 400 μM PI(4,5)P for 45 min to activate the channel, followed by the addition of 1 mM
2
menthol and further incubation for 30 min before vitrification. CryoEM data processing yielded a structure at
3.37 Å resolution in a single conformational state, without imposed symmetry, which we refer to as
TRPV5 , (Supplementary figure 3 and Supplementary table 3).
PIP2+Menthol
The TRPV5 structure reveals a clear density at the intracellular entrance of the pore, positioned
PIP2+Menthol
between the S6 helices, consistent with presence of the menthol molecule (Figure 3a, b). Importantly, this
density was not observed in the previous published TRPV5 structures24,25 (Supplementary figure 4d).
PIP2
The model suggests that one molecule of menthol is primarily coordinated by two W583 residues, forming a
potential hydrogen bond (2.8 Å) between its hydroxyl group and the amide group of W583, as well as a CH-
π interaction (3.3 Å) with the indole ring of the adjacent W583 residue. Notably, W583 has been previously
shown to coordinate the binding of a lysine residue from CaM, which also inhibits TRPV57,34, and to stabilize
the ligand ciss-22a in TRPV635.
Comparing TRPV5 structure with the control structure, TRPV5 in the presence of PI(4,5)P
PIP2+Menthol 2
(TRPV5 )17, we found that the pore remains in a semi-open state (RMSD=0.5 Å) (Figure 3c-i). Specially,
PIP2
the presence of menthol within the pore slightly reduces the pore diameter at I575. This effect is
accompanied by a conformational change in the rotameric angle of W583, which shifts to accommodate
hydrophobic interactions with the ligand. As a result, the pore at W583 adopts a conformation closely
resembling the apo state (Figure 3f). Consistent with this observation, we detected densities at the PI(4,5)P
2
binding site that resemble partially a PI(4,5)P molecule (Supplementary figure 4e). These densities, as
2
previously reported, are in contact with the basic residues R302, R305, K484, and R584, where the
interaction with R584 being one of the most important for inducing the open conformational state12,17,34. Both
our patch-clamp and cryo-EM results suggest that menthol blocks TRPV5 in the open state, a mechanism
like that of CaM, further reinforcing the role of W583 as a key regulatory site for channel modulation.
Menthol binds to W583 in TRPV5 channels.
To functionally validate this interaction, we introduced the W583Q mutation, which removes potential
hydrophobic and aromatic interactions while preserving side-chain size and introducing a polar group. The
W583Q mutation significantly reduced menthol-induced inhibition, shifting the dose-response curve to
higher concentrations (IC = 3.7 mM, Figure 4a and c), indicating that W583 is critical for menthol binding,
50
as suggested by the cryo-EM structure. Single-channel recording confirmed the reduced inhibition by
menthol of this mutant. W583Q channels exhibited distinct gating behavior comparted to WT. Whereas WT
TRPV5 opens in bursts, W583Q channels displayed continuous opening and closing events (Supplementary
Figure 5), similar to the previously reported W583L mutation14. The closed-time distribution contained two
time-constants that remined unaffected by menthol, while the open-time distribution was fitted with two
exponentials components (Supplementary table 1 and 2), consistent with Scheme I, indicating that the
mutations did not greatly alter channel gating. W583Q showed a slight reduction in the slow component with
increasing menthol concentrations. At the highest concentrations, in contrast to WT channels, menthol
reduced the single-channel current amplitude in W583Q, suggesting that in the absence of a tryptophane at
position 583, menthol behaves as a fast blocker, accounting for the remaining block (Supplementary Figure
5d).
Despite this mutation producing a gain-of-function phenotype, shifting channel open probability and the I-V
curve to more positive potentials (Supplementary figure 1 and Supplementary figure 6, respectively),
W583Q retained the inward rectification characteristic of TRPV5 (Figure 4a and Supplementary Figure 6a).
Furthermore, the mutant was also blocked by the pore blocker ruthenium red (RR) with an IC close to that
50
of WT channels, indicating that the mutation does not alter the pore architecture and that its effect is specific
to block by menthol (Supplementary figure 7).
Menthol block was also tested in the previously characterized W583A mutant, for which a cryo EM structure
exists7 (Figure 4b). Similarly to W583Q, W583A showed reduced menthol block (Figure 4b and c). The
observation that both small or large polar substitutions at position W583 greatly impair menthol binding,
supports the importance of hydrophobic interactions in stabilizing menthol binding.
Our cryo-EM structure further suggests that menthol may be stabilized at its binding site through a hydrogen
bond between its hydroxyl group and the indole of W583 (Figure 4d). To test this interaction, we substituted
W583 by a phenylalanine, which lacks an indole group and cannot form hydrogen bonds. The W583F
mutant exhibited a right-shifted menthol dose-response curve, suggesting reduced affinity (Figure 4e, f).
Similarly, W583Y, which retains hydrophobicity but introduces a hydroxyl group, also impaired menthol
block. These results highlight the requirement for specific indole chemistry of tryptophane at position W583
for menthol coordination (Figure 4e, f).
Since many W583 mutation produce gain of function phenotypes (14 and Supplementary figure 6), as control
we introduced mutations at nearby positions. Mutations M578L and G579A, which are localized in the inner
gate and near W583, yielded inwardly rectifying channels with menthol sensitivity similar to WT, confirming
that the effects W583 substitutions on menthol block are specific and not due to gross alteration of pore
structure (Supplementary figure 8). Importantly, G579A has previously been reported to yield stable
macroscopic currents under divalent-free recording conditions14. In the same study, M578 was not identified
as a major contributor to pore constriction, consistent with our observation that the M578L mutation does not
affect menthol sensitivity (Supplementary figure 8).
As presented in the previous section, our structural and functional data reveal that menthol binds to the
intracellular entrance of the TRPV5 pore, specifically interacting with residue W583 while the pore remains
open. To probe the dynamics of menthol pore occupancy, we ran 500 ns all-atom molecular dynamics (MD)
simulations of the open-state TRPV5
PIP2
structure (PDB: 8FFO), with menthol molecules initially placed
randomly in the aqueous phase. Our results showed that the protein stabilized after ~20 ns (RMSD = 2-4 Å,
Supplementary figure 9e), with minor fluctuations (RMSF ~2 Å) observed at the intracellular entrance of the
pore (Supplementary figure 9f), suggesting that the presence of menthol does not induce substantial
conformational changes in the protein. All four simulations replicas showed menthol entering and occupying
the pore from intracellular site (Supplementary figure 9a and b). Trajectory analysis revealed that menthol
molecules can permeate the plasma membrane (Supplementary figure 9d). No Na+ permeation events
occurred during any menthol-occupied state. To quantify the position of menthol relative to the pore, we
measured its distance from I575 (Supplementary figure 9a, b). Unexpectedly, menthol remained at W583 for
only a brief period, forming transient hydrogen bonds lasting 0.1-0.5 ns, before establishing an interaction
with other residues inside the pore, particularly I575, which appeared to stabilize menthol. Once a menthol
occupied the pore, additional menthol molecules interacted more frequently with W583 (Supplementary
figure 9c), suggesting that both W583 and I575 are key residues involved in menthol-mediated TRPV5
blockade.
Effect of mutations at position I575.
To investigate the role of I575 in menthol binding, as suggested by the MD simulations, we tested the I575S
mutant, which should eliminate hydrophobic interactions and introduce a polar residue. This mutation also
diminished menthol inhibition, shifting the dose-response curve to higher concentrations and decreasing the
fraction of blocked current, even at high menthol concentrations (Figure 5a, d, e). The magnitude of this
effect was comparable to that observed in W583Q, suggesting that menthol interacts with I575, or
alternatively, that the presence of a polar residue at position 575 disrupts menthol interactions with W583.
As a control, we tested the I575L mutation, which preserves a hydrophobic residue but could alter sidechain
packing near the protein backbone and should induce little deformation. This mutant retains the menthol
blocking affinity of WT (Figure 5b, d, e). The results indicate that menthol might move from its W583 binding
site to a deeper position within the pore and requires a hydrophobic amino acid at position 575.
Finally, we examined the W583Q-I575S double mutant. These channels remained inwardly rectifying and
retained their affinity for the pore blocker ruthenium red (Supplementary figure 7), indicating that the
combined mutations did not drastically alter TRPV5 gating. However, the double mutant exhibited
significantly reduced susceptibility to menthol inhibition (Figure 5c, d, e), almost completely eliminating block
by menthol, confirming that both W583 and I575 are essential for menthol binding and TRPV5 blockade.

Methods
Molecular biology and cell transfection
The gene encoding full-length rabbit TRPV5 (rbTRPV5; UNIPROT id: Q9XSM3·TRPV5_RABIT) ion channel
cloned in pCINeo/IRES-GFP was a gift from Dr. Tibor Rohacs (Rutgers University). This is a bicistronic
plasmid containing both the channel and a GFP (UNIPROT id: Q17105_AEQVI) sequence under separate
promotors, allowing identification of transfected cells by looking for GFP-fluorescent cells. Human TRPV5
(UNIPROT id: Q9NQA5·TRPV5_HUMAN) was subcloned in the pCDNA3.1(+) vector and was a gift from Dr.
Sharona Gordon (University of Washington). TRPV6 (UNIPROT id: Q9H1D0·_HUMAN) was cloned from
human placenta and was donated to us by Dr. Tibor Rohacs54. Residue numbers refer to the human TRPV5
sequence (729 amino acids); the corresponding regions in human TRPV6 (725 aa) and rabbit TRPV5 (730
aa) are conserved, so numbering is the same across all three proteins in this study.
Single and double mutations were introduced into the rbTRPV5 gene using mutagenic oligonucleotides in
whole-plasmid PCR reactions employing KOD Hot Start DNA polymerase (Novagen-Millipore Corp.)
according to the manufacturer's instructions. PCR products were treated with 1 μl DpnI restriction enzyme
(New England Biolabs) and incubated 5 hrs at 37 °C to remove the parent plasmid. The samples were
transformed into DH5α E. coli competent cells. For each mutant, five colonies were picked and inoculated in
5 mL LB medium, and the plasmids were extracted using a home-made miniprep procedure55. Mutants were
confirmed by automatic sequencing.
Plasmids encoding hTRPV5, hTRPV6 and rbTRPV5-IRES-GFP wild-type and with mutations were
transfected into Human Embryonic Kidney 293 (HEK293) cells (ATCC CRL-1573) using the JetPei reagent
(Polyplus Transfection, France) according to the provider's protocol. Cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine
serum and 1 % of antibiotics (penicillin and streptomycin) (Invitrogen USA), at 37°C, in a 5 % CO
2
atmosphere and were reseeded every 3 days using 0.05 % trypsin-ethylenediaminetetraacetic acid
(Trypsin-EDTA) in 35mm culture dishes over glass coverslips for subsequent electrophysiology assays.
Experiments were performed 19–24 hours after transfection with 1 μg of rbTRPV5-IRES-GFP (wild-type or
mutant). Due to the low cell viability observed after expression of most mutants, a separate plasmid
encoding pEGFP-N1 was introduced to enhance the identification of transfected cells. For single-channel
recordings, 25 ng of wild-type or mutant DNA for 24 hours was used, along with 100 ng of the pEGFP-N1
plasmid, to identify transfected cells.
Patch-clamp recording and data analysis
For patch-clamp recordings, transfected cells were identified as green-fluorescing cells using an inverted
microscope (Nikon Ti) with LED-based fluorescence illumination (478 nm excitation, 505 nm dichroic
mirror, 535/40x nm emission filter) and a 40x objective. Macroscopic currents from HEK293 cells expressing
wild-type or mutant channels were obtained in the outside-out configuration, employing the following
divalent-free extracellular (pipette) and intracellular (bath) solutions (in mM): 140 NaCl , 10 EGTA (ethylene
glycol-bis(β aminoethyl ether)-N,N,N′,N′-tetraacetic acid), 10 HEPES (4-(2-hydroxyethyl)−1
piperazineethanesulfonic acid); intracellular (bath); 140 NaCl, 10 EGTA, 10 HEPES, adjusted to pH 7.2 with
NaOH. Menthol or recording solution was applied by placing the outside-out patch in front of a gravity-feed
perfusion system. All chemicals for solutions were acquired from Sigma-Aldrich. Recording pipettes were
pulled from borosilicate capillary glass (Sutter Instruments) using a P-97 puller (Sutter Instruments) and fire
polished to a final resistance in recording solutions of 3-5 MΩ. Currents were elicited by voltage-clamp
pulses from -100 mV to 50 mV of 200 ms duration applied every 3 seconds and monitored for several
minutes during menthol application. Current-voltage (I-V) curves were obtained by changing the membrane
potential in 10 mV steps from -150 to +100 mV (200 ms duration) every 3 seconds at a 0 mV holding
potential.
Currents were normalized to the initial current at -150 mV to obtain I/I and then averaged. To obtain
max
dose-response curves, currents recorded at -100 mV at each concentration of (-)-menthol, were normalized
to the initial current obtained without the compound and in the presence of 1.5 % ethanol. Dose responses
are plotted as the fraction of blocked current vs. menthol concentration, [menthol] and the dose-response
curves were fitted to Hill equation:
𝑛𝐻 I
𝐼 1
=( ) (1)
𝐼𝑚𝑎𝑥 1+ [𝑚𝑒𝑛𝑡ℎ𝑜𝑙]
𝐼𝐶50
where IC is the half-inhibitory concentration and n is the Hill coefficient.
50 H
Single-channel currents were also recorded from outside-out patches with pipettes having 7-8 MΩ
resistance. Continuous recordings of at least 1 minute duration at -100 mV were obtained before and after
the application of menthol to the outside of the patch. Three menthol concentrations were applied to the
same patch separated by washing in recording solution. Data were filtered at 3 kHz and sampled at 50 kHz
with an EPC 10 patch-clamp amplifier (HEKA Elektronik) run by Patchmaster software (HEKA Elektronik).
Open and closed dwell times were detected employing the 50 % threshold crossing technique56. Dwell time
histograms were compiled and plotted using the logarithmic transform of57 and fitted to sums of equally
transformed exponential functions using a least-squares method to determine time constants associated
with opening, closing and blocking events. Events shorter than the filter dead time, d of 63 μs at 3 kHz,
t
were excluded from the histogram and not considered for fitting. Single-channel analysis was performed
with custom-written software. The voltage-dependance of the single-channel open probability P (V), was fit
o
to the following Boltzmann function:
1
𝑃 (𝑉)= (2)
𝑜 1+𝑒𝑞(𝑉−𝑉0.5)/𝐾𝐵𝑇
Here, q is the apparent gating charge in elementary units, V is the applied potential, V the voltage of half-
0.5
maximal P and K is Boltzmann constant and T the temperature in Kelvin (room temperature 23 oC).
o B
The open and slow mean open time constant data was fitted to the empirical equation:
[𝑚𝑒𝑛𝑡ℎ𝑜𝑙]
(− )
𝜏[𝑚𝑒𝑛𝑡ℎ𝑜𝑙]=545−380∗(𝑒 0.009 𝑚𝑀 ) (3)
Due to reduced expression levels of several mutant channels macroscopic currents were recorded in the
whole-cell configuration. On the day of recording, cells were briefly detached using Trypsin-EDTA and
reseeded onto glass coverslips so they did not attach strongly to the glass surface. After obtaining the
whole-cell configuration, cells were lifted and placed in front of the perfusion system for the application of
different concentrations of menthol or ruthenium red. Single-channel currents from mutant channels were
recorded using the same procedures for WT channels.
Preparation of menthol and ruthenium red solutions
(-)-Menthol was obtained from Santa Cruz Biotechnology, dissolved in absolute ethanol to form a stock
solution at 200 mM, and then diluted from this stock in recording solution at the desired concentration (1 M,
10 M, 100 M, 500 M, 1 mM, 3 mM). These working solutions were then sonicated for 5-10 minutes
before use and prepared fresh for every recording day. The higher concentration of (-)-menthol used (3 mM)
had a final ethanol concentration of 1.5 %, thus, 1.5 % ethanol in recording solution was used as a control in
all experiments after (-)-menthol application and it was determined that it did not affect channel currents.
Ruthenium red (Ammoniated Ruthenium Oxychloride, Sigma-Aldrich) was prepared as a stock solution at
100 M in water. Dilutions where prepared from this stock to reach concentrations of 0.5 nM, 1 nM, 10 nM,
100 nM, 500 nM, 1 M.
Igor Pro 8 software (Wavemetrics) was used for analysis, including fitting, single-channel analyses,
statistical analysis and figure preparation. Data were assumed to follow a normal distribution, and
hypothesis testing was performed using unpaired, two-tailed Student’s t-tests.
TRPV5 protein preparation
TRPV5 preparation and purification followed the protocol described in34. Briefly, the rabbit TRPV5 with a C-
terminal 1D4 epitope tag in a YepM vector 23 was transfected into BJ5457 Saccharomyces cerevisiae
(ATCC) using an alkali-cation yeast transformation kit (MP Biomedicals), according to the instructions of the
manufacturer. Transformed cells were plated on SD-Leu agar plates and allowed to grow at 30°C for 2
days. 30-50 colonies were picked from a plate and inoculated into a single 125 mL starter culture containing
SD-Leu media and 10% v/v glycerol. The culture was incubated at 30°C until reaching the top of the log
phase (OD 1.0–1.4). After confirmation of protein expression by Western blot, the cells were grown at 30°C
with shaking at 200 rpm in 2.5 L of media per flask until OD of 1.0–1.4 was reached. Cells were harvested
by centrifugation at 3000 × g, resuspended in storage buffer (25 mM Tris-HCl, pH 8.0, 300 mM sucrose, and
1 mM PMSF) on ice, and then spun down at 3000 × g to discard the supernatant. Cell pellets were
subsequently stored at −80°C. This expression culture was propagated using 15 mL of the previous growth
at OD 1.0–1.4 for several days until the desired amount was obtained.
Approximately 60 L of growth cells were thawed and resuspended in 300 mL of homogenization buffer (25
mM Tris-HCl, pH 8.0, 300 mM sucrose, 5 mM EDTA, and protease inhibitor cocktail). The resuspended cells
were lysed using a M110Y microfluidizer (Microfluidics) at 100-120 psi. The membranes were separated
from cellular debris by centrifugation at 3,000 × g for 10 min, followed by centrifugation of the supernatant at
14,000 × g for 35 min. The membranes obtained from the previous spin were then pelleted at 100,000 × g
for 1 h. The pelleted membranes were harvested and resuspended on ice using a 15 mL tissue
homogenizer (Kontes Duall with PTFE pestle) in buffer containing 25 mM Tris-HCl, pH 8.0, 300 mM
sucrose, and 1 mM PMSF, and stored at −80°C.
TRPV5 was solubilized from the thawed membranes in solubilization buffer (20 mM HEPES, pH 8.0, 150
mM NaCl, 2 mM TCEP, and 0.87 mM LMNG). The insoluble fraction was separated by centrifugation at
100,000 × g for 1 h. The resulting supernatant was incubated with 1D4-antibody coupled to CnBr-activated
Sepharose beads for 3 h. The beads were collected on a gravity flow column, washed with wash buffer (20
mM HEPES, pH 8.0, 150 mM NaCl, 2 mM TCEP, and 0.064 mM DMNG). The beads were incubated with
elution buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM TCEP, and 0.064 mM DMNG, 3 mg/mL 1D4
peptide) overnight. TRPV5 was eluted in 1 mL fractions every 5 minutes. Peak fractions were pooled and
concentrated using a 100-kDa concentrator (Millipore). For reconstitution into nanodiscs, an equimolar
amount of MSP2N2 protein was incubated with the detergent-solubilized TRPV5 in the presence of soy
polar lipids and DMNG detergent in a 1 mL reaction volume. The molar ratio of TRPV5:MSP2N2:lipid:DMNG
in mixture was 1:1:200:500. Lipids and DMNG were dissolved in 20 mM HEPES, pH 8.0, 150 mM NaCl, 2
mM TCEP (SEC buffer). The reaction mixture was incubated on ice for 30 min, followed by the addition of
~30 μL of Bio-Beads. The mixture was rotated at 4°C for 1 h. The supernatant was transferred to a new
tube, and another 30 μL of fresh Bio-Beads was added before rotating at 4°C overnight to complete the
reaction. Reconstituted TRPV5 was subjected to a Superose 6 increase 10/300 GL column (GE Healthcare)
equilibrated with SEC buffer. Fractions containing nanodisc reconstituted TRPV5 were pooled and
concentrated to 2.57 mg/ml.
Cryo-EM sample preparation and data collection
TRPV5 sample was incubated with 400 μM diC8-PI(4,5)P (Echelon Biosciences, Cat# P-4508) for
PIP2+Menthol 2
45 minutes and 1 mM Menthol (Sigma-Aldrich, Cat# 2216-51-5) for 30 minutes, corresponding to a 1:53:132
(TRPV5:PIP2:Menthol) molar ratio. Menthol was solubilized in 100% ethanol (200 mM) and then diluted at
15% in SEC buffer (30 mM). Final sample contained 1% ethanol. 3 μL TRPV5 sample was applied
PIP2+Menthol
to glow-discharged 1.2/1.3 Quantifoil Holey Carbon Grids (Quantifoil Micro Tools) using a Vitrobot Mark IV
(Thermo Scientific). The sample was blotted in a chamber at 4°C and 100% humidity for 6 s at 0 blotting
force before being freezing in liquid ethane.
Samples were imaged on a Titan Krios G3i 300 kV electron microscope with a Gatan K3 direct electron
detector. 40-42 frame movies were collected with a nominal dose of 41-43 e−/Å2. Super-resolution images
for all datasets were collected at 105,000 magnification with a pixel size of 0.415 Å/pixel.
Data processing
Two datasets of 5,427 (DSA) and 4,736 (DSB) movies were collected and processed in cryoSPARC
v4.4.158,59. Movies were patch motion corrected with a Fourier-crop factor of 0.5 and an alignment resolution
of 3 Å, followed by the patch CTF estimation. Micrographs were manually curated based on a maximum
CTF fit resolution of 8 Å. The template for autopicking were created with 94,896 (DSA) and 94,858 (DSB)
particles blob picked from subsets of 300 micrographs from each dataset and used for extraction. 1,281,269
and 1,491,899 particles were extracted and binned by a factor of 4 with a box size of 72 pixels from DSA
and DSB, respectively. These particles were 2D classified with 100 classes, resulting in 360,885 (DSA) and
225,211 (DSB) good particles that were re-extracted. A three-class heterogeneous refinement was
performed to separate good particles with the TRPV5 apo map (EMD-25716
[https://www.ebi.ac.uk/emdb/EMD-25716]) as a reference, resulting in a good class of 233,564 (DSA)
and 160,664 (DSB) particles at 0.83 and 1.66 Å/pix, respectively. Particles were refined with a non-uniform
refinement with no symmetry to generate the initial model12. Particles from DSA were subjected to 3D
classification with 5 classes focusing on the pore region. The best class, totaling 57,516 particles, were
combined to produce a map with a resolution of 3.53 Å with no symmetry at 0.83 Å/pix. These particles were
subjected to two rounds of heterogeneous refinement and non-uniform refinement, resulting in 33,086
particles, which produced a structure with a resolution of 3.44 Å. Similarly, 22,633 particles were obtained
from DSB after subjected to two rounds of heterogeneous refinement and non-uniform refinement: these
particles generated a structure with a resolution of 3.83 Å. Both datasets were combined and
heterogeneously refined into three volumes. The best structure form with 42.348 particles was refined with a
non-uniform refinement to produce the final map with a resolution of 3.37 Å with no symmetry.
The local resolution was determined in cryoSPARC, and the angular distributions were generated with the
UCSF pyem package.
Model building
The TRPV5 model (8FFO [https://doi.org/10.2210/pdb8FFO/pdb]) (Lee et al. 2023) was used as the
PIP2
initial model and manually adjusted in ISOLDE60. Lipids, PI(4,5)P (PIO), and Menthol (IT9), obtained from
2
the ePDB database, were docked and manually adjusted in COOT61. Real Space_Refinement and eLBOW
from the PHENIX software62 were used to refine and to generate ligand restraints, respectively.
Pore profiles were determined using HOLE63. Chimera, ChimeraX, and PyMol were used for analysis,
visualization and figure generation.
Molecular dynamics simulations
The TRPV5 coordinate model (PDB ID: 8FFO [https://doi.org/10.2210/pdb8FFO/pdb]) (Lee et al. 2023),
PIP₂
oriented using the 6DMU structure from the Positioning of Proteins in Membranes (OPM) server
(http://opm.phar.umich.edu/server.php), was embedded in a 1-palmitoyl-2-oleoyl-sn-glycerol-3-
phosphocholine (POPC) lipid bilayer (606 molecules) along with phosphatidylinositol-4,5-bisphosphate
(model with PIO, 4 molecules) and ergosterol (8 molecules) at pH 7.3. The protein-membrane complex was
solvated with TIP3 water, 100 mM NaCl, and approximately 8 mM menthol, resulting in a system containing
84,087 water molecules, 203 Na⁺, 151 Cl⁻, and 20 menthol molecules within a 160 × 160 × 160 Å³
simulation box (Supplementary Table 4). The membrane-protein system was built using the CHARMM-GUI
membrane builder65 (http://www.charmm-gui.org). The topologies for menthol and PIO were generated
using CGenFF server (https://cgenff.com, Supplementary information), then converted from CHARMM
CGenFF toppar stream files to GROMACS format using cgenff_charmm2gmx_py3_nx2.py
(https://mackerell.umaryland.edu/charmm_ff.shtml) together with the charmm36-jul2022.ff force field
(https://mackerell.umaryland.edu/charmm_ff.shtml). Menthol molecules were placed randomly in the
aqueous region.
Molecular dynamics (MD) simulations were performed using the GPU-accelerated GROMACS 2024.366
package with the CHARMM36m force field67. The system was energy-minimized and equilibrated using the
steepest descent algorithm in CHARMM-GUI without modifications in configuration files (Supplementary
information). In the final equilibration step, the system remained stable with a total energy of −2.75 × 10⁶ ±
691.63 kJ/mol, where the potential energy was −3.76 × 10⁶ ± 1340.85 kJ/mol and the kinetic energy was
1.01 × 10⁶ ± 1171.37 kJ/mol. The temperature remained at 303.18 ± 0.35 K, and the volume at 3631.97 ±
1.17 nm³. The protein RMSD remained at 0.058 ± 0.008 nm. The temperature was maintained at 303 K
using a Nosé−Hoover thermostat, and the pressure at 1 bar using a Parrinello−Rahman barostat. Bond
constraints were applied using the LINCS algorithm. Long-range electrostatic interactions were calculated
using the Particle Mesh Ewald (PME) method with a 12 Å cutoff and updated every 2 fs. Van der Waals
interactions were switched at 10 Å with a 12 Å cutoff. Production simulations consisted of four independent
500 ns runs at 0 mV membrane voltage, and trajectory analyses were performed using GROMACS tools
and VMD.
Molecular docking
Docking of menthol into the cryoEM structure was carried out with AutoDock Vina implemented in
SwisDock68. We used a box of 18 x 18 x 8 Å comprising the position of W583. Default parameters were
used with an exhaustivity of 64.
Data availability
All cryo-EM data generated in these studies are available upon request from Vera Y. Moiseenkova-Bell
(vmb@pennmedicine.upenn.edu). The cryo-EM maps have been deposited in the Electron Microscopy Data
Bank (EMDB) under accession code EMD-70173 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-70173]
(TRPV5 ); EMD-25716 [https://www.ebi.ac.uk/em db/EMD-25716] (TRPV5 ); EMD-29049
PIP2+Menthol Apo
[https://www.ebi.ac.uk/emdb/EMD-29049] (TRPV5 ). The atomic coordinates have been deposited in the
PIP2
Protein Data Bank (PDB) under accession code 9O6G [https://doi.org/10.2210/pdb9O6G/pdb]
(TRPV5 ); 8TF2 [https://doi.org/10.2210/pdb8TF2/pdb] (TRPV5 ): 8FFO
PIP2+Menthol Apo
[https://doi.org/10.2210/pdb8FFO/pdb] (TRPV5 ). All molecular dynamics trajectories can be accessed on
PIP2
Zenodo [https://doi.org/10.5281/zenodo.18472580]69. All other data is included in the paper. Source data are
provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by Méndez-Reséndiz and colleagues reports that menthol is an inhibitor of the Ca-selective TRP channel\nTRPV5. Using macroscopic and single-channel recordings, the authors first find that menthol inhibits in a manner that is\nconsistent with it being a slow channel blocker. The authors then collect cryo-EM images of rabbit TRPV5 in the presence of\nmenthol and PI(4,5)P2, resolving a single structure in which a small non-protein density is observed near the intracellular\nentrance of the pore, where it could act as a pore blocker. This non-protein density is assigned as menthol as it was not\nobserved in previous reconstructions of TRPV5 in the absence of menthol. The modelled menthol interacts with the side\nchains of W583 from each protomer. In contrast to the pose observed in the cryo-EM analysis, Molecular dynamics\nsimulations suggested that menthol binds primarily near I575. Electrophysiological analysis of mutant channels indicated\nthat some mutations to I575 and W583 can disrupt menthol binding. Although the finding menthol\ncan inhibit TRPV5 is\nintriguing, the evidence supporting the proposed mechanism of inhibition is insufficient for publication.\nComments.\n1. It is not clear that the features are sufficiently well resolved in the cryo-EM map made available to the reviewers to model\nthe interactions between TRPV5 and menthol. The densities corresponding to W583, which are proposed to interact with\nmenthol, are poorly resolved, limiting the authors ability to precisely model their positions. The density for the putative\nmenthol is similarly poorly resolved. Collectively, these features limit the authors ability to determine how menthol binds with\nthe channel. Higher quality reconstructions in which the side chains and menthol can be precisely modelled would greatly\nstrengthen this study. This is especially critical as the MD simulations are not in agreement with the binding pose observed\nin the cryo-EM map.\n2. As a technical question, what besides the menthol drives the asymmetry of the reconstruction such the menthol can be\nmodelled in a single pose in which it interacts uniquely with each of the four TRPV5 protomers. The methods did not\ndescribe the use of symmetry expansion, which might aid in defining any asymmetry in the channel-menthol complex.\n3. It is surprising that the menthol is so dynamic in the simulations when it appears to be stably bound in the cryo-EM map.\nAlso, it preferentially binds to residues such as I575 and G579. These interactions do not agree with the cryo-EM analysis,\nwhere a single pose is resolved for menthol bound to W583. It is difficult to reconcile the findings of the two approaches.\n4. Menthol is proposed to act a channel blocker. Was channel activity evaluated during the MD\nsimulations? Does menthol\neffectively block ion permeation in all of the observed poses? What is the effect of membrane potential in MD simulations?\nThe macroscopic recordings appear to indicate that the menthol block is not voltage dependent. A more detailed evaluation\nof the effect of menthol on ion permeation would be helpful in elucidating the mechanism of inhibition.\n5. The lack of agreement between the cryo-EM analysis and the MD simulations makes it difficult to interpret the\nmutagenesis. The authors show that menthol is a less potent inhibitor of the I575S and W583Q mutants, as well as the\nI575S/W583Q double mutant. However, these mutations alter the hydrophobicity and surface potential of the pore. The\nauthors indicate that at least one of these mutants alters channel properties as well. The effects of alternative substitutions at\nthese positions should be tested to better understand the role of I575 and W583 in channel function and menthol binding. It\nis also important to examine other residues near the intracellular gate that are not predicted to interact with menthol.", "reviewer_2": "The study by Mendez-Resendiz and De Jesus-Perez et al. investigates the TRPV5 ion channel, a member of the transient\nreceptor potential (TRP) vanilloid family, and identifies menthol as an inhibitor. This research integrates functional data\nobtained through electrophysiology experiments with structural data from cryo-electron microscopy, and validates binding\nsites via mutagenesis and molecular dynamics (MD) simulations.\nOverall the paper is well written and interesting, although it is not quite clear to me in its current form how far beyond the\nTRP(V) family the general interest of these findings will extend since a clear introduction to the motivation (and potential\nphysiological role) of menthol as a TRPV5 inhibitor is currently missing. That said, I think the paper is publishable after\nclarification of a few details:\nMost importantly, the motivation behind testing menthol as a modulator for TRPV5 function should be elaborated on.\nAdditionally, any physiological context where menthol inhibition of TRPV5 could be relevant, particularly in the kidney,\nshould be discussed. Understanding the rationale behind this study extends beyond developing a pharmacological\ncompound to its broader implications. Furthermore, the introduction lacks the comparison to previous studies looking at\nmenthol as a modulator of e.g. TRPV1 and TRPV3 activity. This becomes more pertinent as these channels lack the central\ntryptophan residue identified here for TRPV5 to be crucial for menthol binding.\nMaterial and Methods :\nPlease specify UniProt or NCBI identifiers for the protein/genes used.\nMaterial methods, line 200, please specify if these are molar ratios. Material methods, line 207. Please indicate the molar\nratios between TRPV5, PIP2, and menthol in your sample preparation. Also, please specify the buffer solutions that your\nrespective additives were dissolved in.\nFor cryo-EM sample preparation, it looks as if you went right for a menthol/PIP2 bound co-complex structure, but you do not\nactually clarify in the introduction the role of PIP2 on TRPV5. So it would be nice to add a couple of sentences on the role of\nPIP2 for TRPV5 activity and why you decided to determine a co-structure rather than a TRPV5 menthol only structure.\nI am unclear about the starting structure(s) of the MD simulation, because currently it reads as if you had only used the prior\nstructure of Lee et al of PIP2-bound TRPV5, which were then adjusted by adding menthol to the simulation. However, what\nabout your newly determined structure in the presence of menthol and PIP2? Is the menthol bound state stable in\nsimulations using your own cryoEM structure as a starting model?\nPlease indicate all equations used for fits (especially referring to Fig 2/S1), either in the material methods or in the\nsupplementary section. Line 459, where is this equation of tau (menthol) and the numbers used therein coming from?\nResult/Discussion\nFig 1/Supplementary Figure 1 shows clear differences for human TRPV5 and V6 compared to rabbit V5, e.g. Hill coefficient\nof ~0.5 and ~1, respectively. A Hill coefficient of <1 indicates negative cooperativity, while the nH of ~1 for rabbit V5 is in\nagreement with the shared binding site in the cryoEM data. This would suggest fundamentally different interaction and/or\nregulation across channels and species. Could you please clarify your models for menthol binding and functional regulation\ncompared between the three proteins? Furthermore, maybe the authors could have another look at the quality of the fits in\nthe supplementary figure and try out different binding models, it seems the current fits are not ideal.\nIn the patch clamp experiments, menthol was added from the outside, but the binding site in the channel seen by EM is on\nthe cytosolic side. Figure 3H places menthol in a solvent channel but interacting with 4 Trp residues. Can the authors please\nadd a few sentences about diffusion of menthol across bilayers and hydrophobicity of this molecule? This is interesting\nbecause menthol is considered to be relatively hydrophobic, but the binding site looks as if it would be accessible from the\nsolvent phase.\nDo the contacts drawn in Fig 3G for the menthol hydroxy group reflect the stabilizing contacts of this molecule? It seems\nlikely that rather than with the indole ring moiety, the OH group might form an H-bond with the NH group of the Trp sidechain\nplaced “above” the menthol? The authors may also possibly better illustrate the hydrophobicity of the binding pocket by a\nspace filling model of both side chain and ligand, but that is just for demonstration purposes.\nMenthol is an asymmetric molecule, interacting with a (presumably) symmetric homotetramer. I feel the authors have missed\nan opportunity to maybe discuss the putatively imposed asymmetry by a ligand in a little more depth.\nThe van der Wijst lab has previously proposed that the tryptophan residue is also important for ion coordination (see e.g.\nhttps://www.pnas.org/doi/10.1073/pnas.1820323116), is menthol inhibition/binding altered in the presence of higher cation\nconcentrations? Furthermore, a W/A mutation shows a gain of function phenotype, it looks as if the W/Q mutant used here\nalso has this effect, but this is not explicitly mentioned in the text. This could potentially be interesting as glutamine, in\ncontrast to alanine, should retain some ion complexation ability? So does this mean that cation complexation at this site is\nnot important for channel regulation?\nI wonder whether the W/Q mutant shows a similar or a more restrained GOF effect than the W/A mutant described in the\nliterature. Can the authors comment on why they chose the W/Q mutant?\nResults around Figure 2: It is interesting that the authors find three time constants for menthol concentration-dependent\nclosure of the channel. Can the authors speculate what these time constants actually reflect on a molecular level? And is the\nchange in the differences in tau shown in Figure 2E as the yellow and the blue traces actually in any way significant for the\ninhibition model the authors propose (as these seem to not significantly change with the ligand concentration)?\nMD simulations: How different is the orientation of the Ile and Trp chains identified here for menthol binding between the\nstarting structure by Lee et al and your own cryoEM structure? How mobile are these residues when menthol is present vs\nwhen it is not bound?\nWith MD you find 2 possible menthol binding sites, is there any density at all at the “upper” site in the cryoEM maps? And\nwhat is the chemical nature of the contacts observed for menthol in the two binding sites in the MD simulations? Related to\nthis, if the contacts between menthol and TRPV5 stem from solely from hydrophobic interactions with the Trp and Ile\nresidues, how is there any specificity for menthol at this site?\nDoes mutation of W/Q or I/S in silico shift the menthol occupancy to the respective “other” binding site? This is a relatively\nstraightforward experiment that would strengthen the relevance of the two binding sites (see also next point):\nWhat was the rationale behind creating an Ile to Ser mutation? I575 is a pore constriction residue in TRPV5, so wouldn’t it\nbe expected that loss of this hydrophobic residue results in loss of menthol block? This may just reflect a more open pore,\nbut does not necessarily mean this residue is in direct contact with menthol, something that the lack in electron density for\nthis site also alludes to?\nMinor points:\nThroughout the introduction, residue numbers are mentioned, presumably following human nomenclature, but possibly also\nrabbit as this is the channel used for this study? This needs specification as the introduction discusses TRPV5 channels\nbroadly and does not distinguish between species.\nFigure 2, it may be my PDF version, but the quality, especially of figure 2A, is quite poor. There are boxes around the plot.\nMaybe the authors could check that.\nLegend Figure 2E and F: I have stumbled a little bit over the phrasing, “the dependence of the three…” (in E) and\n“dependence of the two time constants” (in F). It would help to really clearly write (in the main text and/or the legend)\nwhich time constants you are referring to here, respectively.\nLine 456, spelling mistake in ‘concentration’.\nDetail the preparation of the menthol solution in Ephys experiments, including concentration, solvents used and which\ncontrols were used (it’s not clear to me whether the control was just buffer or contained ethanol?).\nFor legends of figures showing Ephys data, specify that this involves TRPV5 in Hek293 cells.\nAlthough a prior structure of TRPV5 with PIP2 exists, the authors should at least briefly describe the lipid binding site(s) they\nsee in their cryoEM structure in the main text. Do menthol and PIP2 binding affect each other in any way?", "reviewer_3": "In the work of A. Mendez-Resendiz et al. based on data from electrophysiological experiments, determination of the spatial\nstructure of the TRPV5 channel by cryo-EM, point mutagenesis, and molecular dynamics (MD) simulations, it was concluded\nthat menthol blocks the protein in the open state. A menthol binding site formed by W583 residues has been identified in the\nlower gate of the channel. It has been suggested that W583 is involved in menthol-mediated TRPV5 inhibition. Given the\nsignificant physiological role of this class of channels, the presence of pathological conditions caused by disorders in their\nwork, as well as the relatively small number of their known inhibitors, the topic seems relevant, and the results are necessary\nboth for understanding the physical foundations of action of the TRP channels and for the rational design of new\npharmacological agents and therapies. At the same time, the interpretation of some crucial data presented in the paper, a\nnumber of arguments of the authors in their favor, as well as the conclusions drawn, raise serious questions and doubts for\nme, which does not allow, in my opinion, to publish the work in its present form.\nThe main critical points are listed below.\nSince the methods and approaches used by the authors are currently quite routine, including in relation to the study of TRPV\nchannels, the scientific novelty of the work lies only in the identification of a new inhibitor of the TRPV5 channel in the open\nstate - menthol molecules. The evidence presented by the authors in favor of this hypothesis raises the following questions:\n1. The attribution of the density observed in the region of W583 in the cryo-EM model to the menthol molecule is not justified.\nThus, the menthol arrangement shown in Fig. 3g relative to the four W583 residues does not reveal the presence of specific\nand strong intermolecular interactions. In particular, the OH group of menthol is directed towards the nearest 6-membered\nring of indole (the distance is 3.3 A, although it is unclear between which atoms), while the methyl group of menthol forms an\nunfavorable contact with the NH group of the indole ring. etc. It can be assumed that MD results reflect the instability of this\nligand arrangement - the population of such MD-states is significantly lower than a number of alternative states near\nresidues I575, A576 and G579. At the same time, the presence of additional electron density in these areas is not observed\nin cryo-EM. This also contradicts with the phrase on page 13: “…as the I575S and W583Q-I575S mutations drastically\nreduced the potency of menthol blocking, indicating that menthol dynamically interacts with these two residues”. In addition,\nsince the channel narrows in the I575 region, it is quite expected that menthol molecules will be located in this place, since\ntheir size does not allow for free diffusion, at least considering the pore radius (Fig. 3f) and the size of menthol. But the\nauthors do not discuss these important issues.\n2. It is well known (see the reference Van Der Wijst (2017) and other sources, e.g., DOI: 10.1073/pnas.1820323116) that\npoint mutations of W583 can seriously affect the functional characteristics of the channel, therefore, the changes observed\nby the authors in the effect of menthol on the conductivity parameters may be due to a whole set of factors, from which the\nrole of menthol needs to be isolated. It seems to be a difficult task, which has not been completely solved in this work.\nI believe that the conclusions drawn in this case are based on an overly simplified picture.\nOther comments.\n- It is unclear exactly how the 20 menthol molecules were positioned in the initial MD configurations - the authors only\nmention that “menthol molecules were placed randomly in the aquatic region of the intracellular compartment”. However,\nit should be borne in mind that nonpolar menthol molecules can effectively interact with the protein residues closest to them,\nlocking themselves in these positions and not reaching the entrance to the channel. However, according to the authors'\nresults (Fig. S4, b), at least 10 menthol molecules were detected in the pore, between residues I575 and W583. How is such\nselectivity achieved?\n- Parameterization of the force field for the menthol molecule was performed using an automated service, without the\nnecessary calibration on simple model systems. Since menthol is poorly soluble in water, the effects of oligomerization in a\npolar environment are possible – these issues need to be clarified in order to understand how adequate the MD results are.\n- When processing the experimental dose-response curves (Fig. 2 c, d; Fig. 4 d, e), the measured profiles are fitted with\nseveral gaussians, although this is not justified in any way. As a result, such a decomposition in many cases looks incorrect\nand, accordingly, distorts the conclusions.\n- Page 10: “The single-channel current amplitude remained unaffected by menthol, as indicated by all-points amplitude\nhistograms of selected current segments (Figure 2b)”. But the histograms are rather different… Moreover, they are different\nfrom those reported for WT TRPV5 in (DOI: 10.1038/srep45489) (Fig. 2f). But these effects are not discussed.\n- The curves in Figure S1 c, e do not reach a plateau, how in this case the Hill coefficient can be estimated?\nIt is unclear exactly what information is needed to understand the conclusions provided by the graphs of RMSF versus MD\ntime (Figure S4, f)? The authors remarked that such plots reveal “minor fluctuations (~2 Å) observed at the intracellular\nentrance of the pore” (page 11). But what was the reason to report the whole RMSF plots without discussing them?\n- Page 12: The meaning of the following phrase is not clear to me: “…in the absence of a tryptophane at position 583,\nmenthol blocks TRPV5 by rapidly entering and exiting the conduction pathway, albeit with lower affinity”."}, "author_response": "We thank the reviewers for insightful and constructive comments. We have carried out most of\nthe suggested changes and answered all the comments included in the three reviews. These\ninclude changes to the manuscript text, new analysis and new experiments. We think that these\nchanges provide further support for menthol being a pore blocker primarily binding to residue\nW583. The manuscript has been strengthened, and we have clarified several sections of it. Below\nwe provide a point-by-point answer to the reviewers’ comments. Our individual answers are\nwritten in blue text and we have indicated new and changed text in our revised manuscript in\ngreen highlight.\nReviewer #1 (Remarks to the Author):\nThis study by Méndez-Reséndiz and colleagues reports that menthol is an inhibitor of the Ca-\nselective TRP channel TRPV5. Using macroscopic and single-channel recordings, the authors\nfirst find that menthol inhibits in a manner that is consistent with it being a slow channel blocker.\nThe authors then collect cryo-EM images of rabbit TRPV5 in the presence of menthol and\nPI(4,5)P2, resolving a single structure in which a small non-protein density is observed near the\nintracellular entrance of the pore, where it could act as a pore blocker. This non-protein density is\nassigned as menthol as it was not observed in previous reconstructions of TRPV5 in the absence\nof menthol. The modelled menthol interacts with the side chains of W583 from each protomer. In\ncontrast to the pose observed in the cryo-EM analysis, Molecular dynamics simulations suggested\nthat menthol binds primarily near I575. Electrophysiological analysis of mutant channels indicated\nthat some mutations to I575 and W583 can disrupt menthol binding. Although the finding menthol\ncan inhibit TRPV5 is intriguing, the evidence supporting the proposed mechanism of inhibition is\ninsufficient for publication.\nComments.\n1. It is not clear that the features are sufficiently well resolved in the cryo-EM map made available\nto the reviewers to model the interactions between TRPV5 and menthol. The densities\ncorresponding to W583, which are proposed to interact with menthol, are poorly resolved, limiting\nthe authors ability to precisely model their positions. The density for the putative menthol is\nsimilarly poorly resolved. Collectively, these features limit the authors ability to determine how\nmenthol binds with the channel. Higher quality reconstructions in which the side chains and\nmenthol can be precisely modelled would greatly strengthen this study. This is especially critical\nas the MD simulations are not in agreement with the binding pose observed in the cryo-EM map.\nWe sincerely thank the reviewer for their thoughtful and constructive evaluation of our cryo-EM\ndata and proposed menthol binding site.\nAs correctly noted, the density corresponding to W583 side chains and the putative menthol ligand\nis not resolved at a level that permits precise atomic modelling of side-chain conformations or\nunambiguous placement of the ligand. We believe that the limited resolution around W583 is not\nincidental, but rather a consequence of the dynamic nature of menthol binding and its\npredominantly hydrophobic interactions. Menthol is a small, hydrophobic molecule that does not\nengage in strong, directional interactions and is unlikely to strongly contact W583 residues from\nevery subunit simultaneously. As such, it does not sufficiently anchor the surrounding residues to\nenable accurate modelling of their side-chain conformations. Furthermore, we emphasize that our\ncryo-EM map was refined without imposing symmetry, which may reduce the accuracy of\naveraged density in this region.\nDespite these limitations, we observe a reproducible non-protein density at the intracellular\nentrance of the pore. This density is absent in control structures and is consistent in size and\nshape with that of menthol, supporting its interpretation as menthol occupancy. Given the\nproximity of this density to W583 and the established functional role of W583 in CaM binding, we\npropose this as a plausible, albeit not definitive, orientation of menthol within the binding site.\nUpon re-evaluation of our data, we observed that menthol may adopt an alternative pose in which\nthe hydroxyl group of menthol forms a potential hydrogen bond (2.8 Å) with the amide group of\nW583, and a CH-π interaction may occur with the indole ring of an adjacent W583 residue (3.3\nÅ). This prompted us to generate and test the W583F mutant, which retains the aromatic π surface\nrequired for hydrophobic CH–π contacts but eliminates the indole N–H hydrogen-bond donor\nunique to tryptophan. As expected, W583F displayed a reduced menthol effect, supporting a\nmodel in which menthol engages W583 through both π-mediated hydrophobic interactions and a\nspecific hydrogen bond to the indole N–H. Similarly, the newly analysed W583Y mutant maintains\nan aromatic ring and has a hydroxyl group, but this hydroxyl group cannot replace the indole N–\nH hydrogen bond present in tryptophan, leading to a similar reduction in menthol’s effect (Figure\n4d-f).\nWhile our molecular dynamics simulations indicate that menthol does remain bound to W583,\nthere are also transient contacts are observed with I575, a residue identified through MD as\nshaping a hydrophobic region and facilitating ligand accommodation. This interpretation is\nsupported by our mutagenesis experiments, which show that both I575S and W583Q mutations\nsignificantly reduce menthol-mediated inhibition, indicating that both residues contribute to\nmenthol’s functional modulation of TRPV5.\nRegarding the molecular dynamics simulations, these were conducted independently of the cryo-\nEM model to explore the dynamics of menthol-channel interactions. While the simulations suggest\nalternative poses and transient interactions with W583, we interpret these as complementary to,\nrather than contradictory of, the cryo-EM data. The differences between the experimentally\nobserved density and the MD-derived poses likely reflect the limitations of both approaches,\nincluding obtaining the structure in its most stable state and the inherent approximations and\ntimescale constraints of MD simulations.\n2. As a technical question, what besides the menthol drives the asymmetry of the reconstruction\nsuch the menthol can be modelled in a single pose in which it interacts uniquely with each of the\nfour TRPV5 protomers. The methods did not describe the use of symmetry expansion, which\nmight aid in defining any asymmetry in the channel-menthol complex.\nAs suggested, we did perform symmetry expansion on the final reconstructed map to investigate\npotential asymmetries in menthol binding. However, using this approach did not improve the\nresolution or clarify additional features related to ligand binding. Specifically, the symmetry-\nexpanded particles did not yield better-defined density for menthol or reveal distinct binding poses\nacross the four protomers.\nGiven these results, we opted to proceed with the conventional (non-symmetry-expanded)\nreconstruction strategy, which provided the most interpretable and reliable density for the\ncomplex. However, as indicated in our response to the previous question, reprocessing of our\ndata suggest specific interactions in which menthol may adopt an alternative pose in which the\nhydroxyl group of menthol forms a potential hydrogen bond (2.8 Å) with the amide group of\nW583, and a CH-π interaction may occur with the indole ring of an adjacent W583 residue (3.3\nÅ) (Figure\n3g and Figure 4d-f).\n3. It is surprising that the menthol is so dynamic in the simulations when it appears to be stably\nbound in the cryo-EM map. Also, it preferentially binds to residues such as I575 and G579. These\ninteractions do not agree with the cryo-EM analysis, where a single pose is resolved for menthol\nbound to W583. It is difficult to reconcile the findings of the two approaches.\nWe thank the reviewer for raising this important point. We cannot say categorically that cryoEM\nresult and MD simulation are not in agreement, but rather, MD gives us additional information\nabout the dynamics of the blocking process. Our cryo-EM data identify W583 as the principal\nmenthol-binding site, while the MD simulations provide complementary information by suggesting\na second, transient interaction near I575. This additional site may be relevant for understanding\nthe electrophysiological recordings, as it reflects the dynamic nature of menthol binding and its\nrelationship to the narrow part of the pore. We also note that the experimental conditions for MD,\ncryo-EM, and electrophysiology differ which likely contributes to the apparent discrepancies.\nUnfortunately, were not able to solve the structure for this second site, but we observed a density\nat this position in a subset of particles, although these were not sufficient to achieve high\nresolution (Figure R2). Taken together, we interpret the MD as an approach that complements\nthe structural and functional data, with W583 representing the main binding site and I575\ncontributing to a more dynamic, secondary interaction.\nAs mentioned above, we acknowledge that there are some factors that may help explain the\napparent discrepancies in menthol binding between the experimental results and our simulations:\nFirst, Force-field limitations for menthol. Menthol parameters were generated using the CHARMM\nGeneral Force Field (CGenFF). Although slightly penalties were reported during\nparameterization, subtle inaccuracies in partial charges or dihedral potentials may reduce the\nfidelity of menthol’s interactions in our system. For example, the dipole moment calculated in a\nsystem with menthol and solution results ~2.1 D, a value slightly higher than published previously\n(1.4 D)1.\nDifferences in experimental versus simulation conditions. Prior to vitrification, the protein was\nincubated at 4 °C with diC8-PI(4,5)P₂ and menthol for 30 minutes, whereas in our MD simulations\nthe system was held at 29.8 °C for 500 ns. This temperature difference corresponds to an\nestimated menthol diffusivity of ~3.7 × 10⁻¹⁰ m²/s at 4 °C versus ~8 × 10⁻¹⁰ m²/s at 29.8 °C. As a\nresult, over a 100-ns trajectory, menthol would sample on average ~4.7 nm at 4 °C and ~7 nm at\n29.8 °C, increasing its likelihood of exploring alternative binding poses in silico. Furthermore, our\nsimulations (∼500 ns) are short relative to the timescales over which experimental equilibrium is\nachieved. We therefore cannot rule out that menthol might converge on the single W583 pose at\nlonger timescales or under different sampling strategies.\nDespite these caveats, our MD results have been instrumental in guiding mutagenesis\nexperiments, as mutations at residues highlighted by the simulations produced the largest shifts\nin TRPV5’s menthol sensitivity, corroborating the biological relevance of our computational\npredictions. For example, although MD suggested that menthol may form transient contacts with\nI575 in the narrow part of the pore, no defined density was observed at this site in our cryo-EM\nmaps. Consistently, the I575S mutation abolished menthol inhibition, most likely due to disruption\nof the local hydrophobic environment rather than loss of a specific binding interaction, whereas\nthe I575L mutation preserved inhibition, indicating that a large hydrophobic side chain at this\nposition is sufficient to maintain pore properties.Together, these results suggest that I575 shapes\nthe hydrophobic context of the pore but may not constitute a primary menthol-binding determinant,\nwhich is more clearly supported by the cryo-EM density and mutagenesis at W583. A comparable\nmechanism has been described for tetra-butyl ammonium (TBA) block of hERG K+ channels,\nwhere the blocker first binds to an aromatic residue in the cavity and then moves further up the\npore to interact with a narrowing isoleucine site2 (discussion, page 10, lines 284-286).\n4. Menthol is proposed to act a channel blocker. Was channel activity evaluated during the MD\nsimulations? Does menthol effectively block ion permeation in all of the observed poses? What is\nthe effect of membrane potential in MD simulations? The macroscopic recordings appear to\nindicate that the menthol block is not voltage dependent. A more detailed evaluation of the effect\nof menthol on ion permeation would be helpful in elucidating the mechanism of inhibition.\nYes, we monitored ion flux throughout the molecular dynamics simulations to assess the impact\nof menthol on channel conduction. Under the conditions simulated, no ion permeation events\nwere observed. While Na⁺ ions were consistently present and coordinated within the selectivity\nfilter and lower vestibule (Figure S9), they did not possess sufficient energy to traverse the lower\ngate of the channel.\nThis absence of conduction is expected given the lack of a driving force in our setup. Specifically,\nthe simulations were performed under symmetric NaCl concentrations and at 0 mV membrane\npotential. Therefore, we chose not to introduce a transmembrane potential in the simulation in\norder to focus on spontaneous binding and occupancy under equilibrium-like conditions.\nImportantly, when menthol occupied the pore, particularly in the position near I575, it effectively\noccluded the conduction pathway. Although some water molecules were able to pass through, no\nion flux was detected, supporting the notion of a physical block. These observations are consistent\nwith menthol acting as a pore blocker and help to illustrate its inhibitory mechanism, even in the\nabsence of transmembrane voltage.\nWe have now clarified these aspects in the revised Results sections to better highlight the\nfunctional interpretation of the MD simulations in the context of our electrophysiological data\n(Results, page 8, lines 218-233).\nFurthermore, in response to the reviewer comment, we performed two replicas of MD simulation\napplying a voltage of −400 mV for 200 ns to determine the effect of membrane potential on TRPV5\nin the presence of menthol. As in our previous simulations without voltage, Na⁺ could enter the\npore and reach its vestibule from the extracellular side (distance region >0 in the Figure R1).\nHowever, no ions crossed the conduction path before menthol entered from the intracellular side.\nLikewise, some cations accessed the pore from the intracellular side (distance region <0), but\nnone passed through, either because the electric field direction was unfavourable or because\nmenthol was already blocking the pore.\nFigure R1. Distances of menthol molecules or sodium ions from the center of mass of I575 were\nmeasured in two MD simulations of 200 ns each, applying a voltage of –400 mV. Negative values\nindicate that molecule occupies the pore from the intracellular side. Dashed lines indicate the\ncenter of mass of I575 and W583. In both simulations, menthol occupied the pore (blue),\nremaining at I575 as observed in the simulations without voltage. Similarly, Na⁺ was able to enter\nthe pore, more frequently from the extracellular side; however, no permeation events were\nobserved.\nIn addition, we thank the reviewer for raising the point about the voltage dependence of channel\nblock by menthol. To address this, we analysed our data to estimate the voltage dependence of\nthe fraction of blocked current at three menthol concentrations. This analysis shows that menthol\nblock is indeed slightly voltage dependent. This result was intriguing since menthol is a neutral\nmolecule and the identified binding site lies outside the membrane electric field, making a classical\nWoodhull mechanism unlikely3. However, the observed voltage dependence can be explained by\na model in which the channel opens after transitions between two closed states and becomes\nblocked only in the open state. The voltage dependence of block arises from the fact that opening\nis voltage-dependent and thus blocking is coupled to channel opening. This linear model, based\non van der Wijst et al.4, was further constrained by measuring the voltage dependence of the\nchannel opening In single-channel recordings (Supplementary fig S1). The model accounts for\nboth the dose dependence and the observed voltage dependence of menthol block. We have\nincluded this data in a new Figure 1 to clarify the mechanism of channel block by menthol and its\neffect on ion permeation, as suggested by the reviewer (Page 4, lines 102-111). Model details are\npresented in an appendix. We have also carried out a characterization of the effect of high\nconcentration of permeant ions (300 mM Na+) on the blocking effect of menthol, as suggested by\nreviewer 2. The result of these experiments indicates that the fraction of current blocked by\nmenthol remains the same for control (140 mM Na+) and high Na+, suggesting that menthol does\nnot compete for a permeant ion binding site (see Figure R2).\nFigureR2. Effect of extracellular Na⁺ concentration on menthol block of TRPV5. a\nRepresentative whole-cell currents recorded at –100 mV in the absence (black traces) or\npresence of 500 µM menthol (blue traces) with 140 mM (continuous traces) or 300 mM (dotted\ntraces) extracellular NaCl. b Representative whole-cell currents recorded at –100 mV in the\nabsence (black traces) or presence (orange traces) of 3 mM menthol with 140 mM (continuous\ntraces) or 300 mM (dotted traces) extracellular NaCl. c Fractional inhibition by 500 µM (blue) and\n3 mM (orange) menthol under 140 mM and 300 mM extracellular Na⁺ conditions. Each symbol\nrepresents an individual measurement.\n5. The lack of agreement between the cryo-EM analysis and the MD simulations makes it difficult\ninterpret the mutagenesis. The authors show that menthol is a less potent inhibitor of the I575S\nand W583Q mutants, as well as the I575S/W583Q double mutant. However, these mutations alter\nthe hydrophobicity and surface potential of the pore. The authors indicate that at least one of\nthese mutants alters channel properties as well. The effects of alternative substitutions at these\npositions should be tested to better understand the role of I575 and W583 in channel function and\nmenthol binding. It is also important to examine other residues near the intracellular gate that are\nnot predicted to interact with menthol.\nWe appreciate the reviewer’s insightful comments regarding the interpretation of our mutagenesis\ndata in the context of the cryo-EM and MD results. We agree that cryo-EM provides a static picture\nwhereas MD highlights the dynamic nature of menthol binding, suggesting transient interactions\nnot only with W583, but also with I575 in the narrow part of the pore. Although no defined density\nwas observed at I575 in the cryo-EM maps, the I575S and I575L mutants help clarify its role: the\nloss of menthol inhibition in I575S is most likely due to disruption of the local hydrophobic\nenvironment rather than elimination of a specific menthol contact, while preservation of block in\nI575L supports the importance of maintaining a hydrophobic side chain at this constriction. By\ncontrast, W583 emerges as the primary determinant of menthol binding, as supported by cryo-\nEM density, MD, and mutagenesis. Substitution of W583 with either phenylalanine or tyrosine\nretained aromaticity but impaired menthol block, demonstrating that the indole NH group and\nextended π-surface unique to tryptophan are essential (Figure 4 and page 7, lines 197-204).\nTo address the reviewer’s concern about nonspecific effects, we have now introduced mutations\nat neighboring residues (G579A and M578L) that are not predicted to interact with menthol. Both\nmutants behaved very similarly to wild type and did not alter menthol block, supporting the\nspecificity of W583 as a binding site (Supplementary figure 8). Furthermore, electropysiology\nconfirmed that all mutants preserved the general channel properties, including conductance–\nvoltage relationships, inward rectification, and blok by ruthenium red, indicating that the observed\neffects are not due to a disruption of pore function (Supplementary figures 6 and 7).\nTogether, these results resolve the apparent differences between cryo-EM and MD by showing\nthat menthol binding is dynamic, with W583 as the principal site and I575 contributing to the\nhydrophobic environment of the pore, while control mutations confirm that the effects are not\nattributable to nonspecific pore alterations.\nReviewer #2 (Remarks to the Author):\nThe study by Mendez-Resendiz and De Jesus-Perez et al. investigates the TRPV5 ion channel,\na member of the transient receptor potential (TRP) vanilloid family, and identifies menthol as an\ninhibitor. This research integrates functional data obtained through electrophysiology experiments\nwith structural data from cryo-electron microscopy, and validates binding sites via mutagenesis\nand molecular dynamics (MD) simulations.\nOverall the paper is well written and interesting, although it is not quite clear to me in its current\nform how far beyond the TRP(V) family the general interest of these findings will extend since a\nclear introduction to the motivation (and potential physiological role) of menthol as a TRPV5\ninhibitor is currently missing. That said, I think the paper is publishable after clarification of a few\ndetails:\nMost importantly, the motivation behind testing menthol as a modulator for TRPV5 function should\nbe elaborated on. Additionally, any physiological context where menthol inhibition of TRPV5 could\nbe relevant, particularly in the kidney, should be discussed. Understanding the rationale behind\nthis study extends beyond developing a pharmacological compound to its broader implications.\nFurthermore, the introduction lacks the comparison to previous studies looking at menthol as a\nmodulator of e.g. TRPV1 and TRPV3 activity. This becomes more pertinent as these channels\nlack the central tryptophan residue identified here for TRPV5 to be crucial for menthol binding.\nWe agree with the reviewer that the rationale for this study extends beyond compound\ndevelopment and lies in understanding how a monoterpene such as menthol differentially\nmodulates members of the TRP family. Menthol is a well-established activator of TRPM8, an\nagonist of TRPV3, and exerts inhibitory effects on TRPV1 and TRPV4, even though these\nchannels lack the central tryptophan residue that we identify here in TRPV5. This comparison\nhighlights the novelty of our work: while menthol’s effects on TRPV1 and TRPV3 involve residues\nin the S4–S5 linker, our results demonstrate that in TRPV5 menthol interacts with W583 at the\nlower gate to function as a slow pore blocker. Thus, our findings uncover a structurally distinct\nand channel-specific mechanism that broadens the understanding of how menthol can exert both\nagonist and inhibitory actions across the TRP channel family. We have now better argued the\nrationale behind our study and improved the presentation regarding the possible physiological\nimplications of menthol block of TRPV5 channels in the introduction (page 3, lines 76-83). We\nhave also moved mention of menthol effects on other TRP channels from the discussion to the\nintroduction as requested (page 3) and improved the discussion of these findings (page 9, lines\n259-269).\nIn addition, we have expanded the Discussion to explicitly address menthol’s effects on other\nTRP channels and to emphasize this conclusion.\nMaterial and Methods:\nPlease specify UniProt or NCBI identifiers for the protein/genes used.\nWe have now included the UNIPROT identifiers for the proteins used un this study. RabbitTRPV5\nchannel UNIPROT identifier: Q9XSM3 · TRPV5_RABIT. HumanTRPV5 channel, UNIPROT\nidentifier: Q9NQA5 · TRPV5_HUMAN. HumanTRPV6 channel, UNIPROT identifier:\nQ9H1D0 · TRPV6_HUMAN. Green Fluorescent Protein (GFP): Q17105 · Q17105_AEQVI.\nMaterial methods, line 200, please specify if these are molar ratios. Material methods, line 207.\nPlease indicate the molar ratios between TRPV5, PIP2, and menthol in your sample preparation.\nAlso, please specify the buffer solutions that your respective additives were dissolved in.\nWe added to Materials and methods\n“The molar ratio of TRPV5:MSP2N2:lipid:DMNG in mixture was 1:1:200:500. Lipids and DMNG\nwere dissolved in 20 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM TCEP (SEC buffer).”\n“TRPV5 sample was incubated with 400 μM diC8-PI(4,5)P (Echelon Biosciences, Cat#\nPIP2+Menthol 2\nP-4508) for 45 minutes and 1 mM Menthol (Sigma-Aldrich, Cat# 2216-51-5) for 30 minutes,\ncorresponding to a 1:53:132 (TRPV5:PIP2:Menthol) molar ratio.”\nFor cryo-EM sample preparation, it looks as if you went right for a menthol/PIP2 bound co-\ncomplex structure, but you do not actually clarify in the introduction the role of PIP2 on TRPV5.\nSo it would be nice to add a couple of sentences on the role of PIP2 for TRPV5 activity and why\nyou decided to determine a co-structure rather than a TRPV5 menthol only structure.\nThe role of PIP2 of TRPV5 is described in the introduction:\n“TRPV5 channels display a marked inward rectification and are constitutively open at negative\npotentials, allowing for passive entry of cations into cells at the resting membrane potential.\nRegulation of TRPV5 is mainly carried out by the control of phosphatidylinositol 4,5-bisphosphate\n(PI(4,5)P2) lipid concentration, which binds between the N-linker (Arg302, Arg305), S4-S5 linker\n(Lys484), and the S6 helix (Arg584) to keep the channel open (Hughes et al. 2018).”\nWe clarify the rationale in the Result section:\n“A priori, it is unclear whether menthol solely blocks the channel or also induces an alternative\nconformational state to close the channel. Given that TRPV5 adopts an open state in the presence\nof PI(4,5)P , we initiated our experiments with a PIP₂-activated channel and subsequently applied\n2\nmenthol to assess its effect” (Page 12, line 341-346).\nI am unclear about the starting structure(s) of the MD simulation, because currently it reads as if\nyou had only used the prior structure of Lee et al of PIP2-bound TRPV5, which were then adjusted\nby adding menthol to the simulation. However, what about your newly determined structure in the\npresence of menthol and PIP2? Is the menthol bound state stable in simulations using your own\ncryoEM structure as a starting model?”\nOur simulations were designed to explore the process of menthol binding and its potential\nmechanism of channel blockade. In line with the rationale behind our experimental design, where\npurified TRPV5 was incubated with PI(4,5)P₂, to open the channel, and menthol, we aimed to\ncapture spontaneous binding events in silico. To this end, and given the extensive computational\nresources required to achieve relevant simulation timescales (~15 ns/day on our setup), we\ninitiated simulations using the previously published structure of TRPV5 bound to PI(4,5)P₂ 6, while\nour cryo-EM data were still being processed.\nBecause the menthol binding site was unknown a priori, menthol molecules were placed randomly\nin the solvent at locations sufficiently distant from the protein to avoid introducing bias in their\npositioning. This allowed us to observe spontaneous interactions and possible binding pathways\nwithout constraining the ligand to a predefined pose.\nWe acknowledge that our newly determined cryo-EM structure provides a snapshot of menthol\nbound at W583. However, given the high mobility of menthol observed in our simulations, even\nafter transient binding to W583 or deeper sites such as I575, we predict that starting from our own\nstructure would not have significantly altered the outcomes. As discussed previously, this dynamic\nbehavior is likely influenced by a combination of factors, including menthol’s hydrophobic nature,\nthe absence of a strong anchoring site, and limitations in the ligand parameterization.\nPlease indicate all equations used for fits (especially referring to Fig 2/S1), either in the material\nmethods or in the supplementary section. Line 459, where is this equation of tau (menthol) and\nthe numbers used therein coming from?\nThanks for pointing out this oversight. We have added the Hill and the Boltzmann equations used\nfor fitting the data to the Methods section and in an appendix with the derivation of the equations\nfor the steady state from Scheme I, which were used to fit the WT TRPV5 data as a well as the\nhTRPV5 and hTRPV6 dose responses in supplementary figure 2. Regarding the equation used\nto fit the open dwell times in figure 2, it does not have any theoretical meaning.\nResult/Discussion\nFig 1/Supplementary Figure 1 shows clear differences for human TRPV5 and V6 compared to\nrabbit V5, e.g. Hill coefficient of ~0.5 and ~1, respectively. A Hill coefficient of <1 indicates\nnegative cooperativity, while the nH of ~1 for rabbit V5 is in agreement with the shared binding\nsite in the cryoEM data. This would suggest fundamentally different interaction and/or\nregulation across channels and species. Could you please clarify your models for menthol binding and\nfunctional regulation compared between the three proteins? Furthermore, maybe the authors\ncould have another look at the quality of the fits in the supplementary figure and try out different\nbinding models, it seems the current fits are not ideal.\nThank you for your comment. We have reanalyzed the hTRPV5 and hTRPV6 menthol blocking\ndata (Supplemental figure 2). Specifically, since these currents were of relatively small amplitude,\nwe have subtracted the leak current, which we realized has a larger effect in patches with less\nchannel-mediated current, and re-calculated the menthol-blocked fraction. The newly analyzed\ndata show a steeper dependence on the menthol concentration and can be better fit to the Hill\nequation, with values of the Hill coefficient, nH, closer to 1, as observed with the rabbit TRPV5\nchannel. Following your suggestion, we have also fitted these data to a linear gating model\nincorporating menthol blocking of the open state that includes two closed and one open states.\nWe now show that this model provides a good fit to several aspects of TRPV5 block by menthol,\nincluding the dose response and the voltage-dependence of block (Figure 1). As can be seen in\nthe new Supplementary figure S2, the model is also able to describe the hTRPV5 and hTRPV6\nmenthol dose response data reasonably well. It must be emphasized that since the model\nconsiders a single menthol binding event, the expected steepness of the dose-response curve is\nequivalent to a Hill coefficient of exactly 1. The deviation of the fitted nH from 1 is within\nexperimental error.\nIn the patch clamp experiments, menthol was added from the outside, but the binding site in the\nchannel seen by EM is on the cytosolic side. Figure 3H places menthol in a solvent channel but\ninteracting with 4 Trp residues. Can the authors please add a few sentences about diffusion of\nmenthol across bilayers and hydrophobicity of this molecule? This is interesting because menthol\nis considered to be relatively hydrophobic, but the binding site looks as if it would be accessible\nfrom the solvent phase.\nThis is an important point to clarify. We have commented in the discussion (page 9, lines 259-\n262) on the fact that menthol is hydrophobic although a better description might be that it has an\namphiphilic character. It has low solubility in water (dipole moment 1.75 D), with a tendency to\nform aggregates at high concentration. Importantly, it has been shown by simulation that menthol\ncan readily partition and cross into phospholipid membranes at low concentrations and thus it is\nphysically possible for it to cross from the outside to the inside of the plasma membrane of cells7.\nMenthol capacity to penetrate cell membranes is often exploited to increase the cell penetration\nof other organic compounds8 and it has been observed by experiment and simulation that at high\nconcentrations menthol can disrupt the lipid organization within phospholipid membranes. Our\nown MD simulations borne these observations. It can be seen in Supplementary figure S9d that\nmolecules of menthol cross from one side to the other of the membrane with relative ease. Several\namphiphilic molecules that bind to TRP channels such as capsaicin also reach membrane\naccessible or intracellular facing sites by crossing the plasma membrane. This has been observed\nboth experimentally and in simulations9.\nDo the contacts drawn in Fig 3G for the menthol hydroxy group reflect the stabilizing contacts of\nthis molecule? It seems likely that rather than with the indole ring moiety, the OH group might\nform an H-bond with the NH group of the Trp sidechain placed “above” the menthol? The authors\nmay also possibly better illustrate the hydrophobicity of the binding pocket by a space filling model\nof both side chain and ligand, but that is just for demonstration purposes.\nWe sincerely appreciate the reviewer’s insightful observation. Following this suggestion, we\nreanalyzed our model and adjusted the pose of menthol relative to W583. In this revised\nconformation, the hydroxyl group of menthol forms a potential hydrogen bond with the backbone\nNH group of W583, which is positioned directly above the ligand. Additionally, the menthol methyl\ngroup appears to engage in a CH–π interaction with the indole ring of an adjacent W583 residue,\nfurther supporting the proposed stabilizing contacts (Figure 3g).\nWe agree that visualizing the hydrophobic nature of the binding pocket could enhance the\nreader’s understanding. In response, we have included a surface representation of the menthol-\nbinding site in the revised figure (Supplementary Figure S4f), highlighting the hydrophobic\nenvironment and illustrating the proximity of menthol to surrounding side chains.\nThese changes have been incorporated into the revised figure and described in the figure legend\nand main text accordingly.\nTo investigate the possibility that menthol might form a hydrogen bond with the NH group of indole\nin tryptophane, we have analyzed the effects if W583F and W583Y mutants. These two mutants\nhave impaired binding to menthol, indicating that an aromatic residue at 583 is not sufficient for\nmenthol binding and strongly suggesting that menthol interacts with indole through hydrogen\nbonding (Figure 4 and page 7, lines 197-204).\nMenthol is an asymmetric molecule, interacting with a (presumably) symmetric homotetramer. I\nfeel the authors have missed an opportunity to maybe discuss the putatively imposed asymmetry\nby a ligand in a little more depth. “\nWe thank reviewer’s suggestion. We discuss more about this point in the Discussion section\n(page 10, lines 292-305):\n“Interestingly, in our structures, TRPV5 bound to PIP₂ and menthol preserves an overall C4\nsymmetry, yet this arrangement is locally perturbed at the ligand-binding site. This contrasts with\nTRPV5 in complex with calmodulin or TRPV6 bound to cis-22, where C4 symmetry is maintained\neven at the W583 binding site. We interpret this localized disruption as a consequence of\nmenthol’s pronounced molecular asymmetry. One terminus of menthol contains a donor/acceptor\nregion capable of forming strong hydrogen-bonding interactions with the NH group of W584, while\nthe remainder of the molecule is densely populated with CH groups, favoring CH–π interactions\nwith tryptophan aromatic rings. The small size of menthol likely prevents simultaneous\nengagement with all four W583 residues, limiting its capacity to induce the large-scale\nconformational rearrangements of the pore observed in other TRP family members, such as\nTRPV2 and TRPM2, which transition to a global C2 symmetry upon activation by RTX or during\nintermediate gating steps, respectively 10,11. Instead, the conformation captured here appears to\nrepresent a hybrid between the canonical PIP₂-bound open state and a partially obstructed state\nin which two adjacent W583 residues are stabilized by menthol in a geometry optimal for impeding\nion conduction.”\nThe van der Wijst lab has previously proposed that the tryptophan residue is also important for\nion coordination (see e.g. https://www.pnas.org/doi/10.1073/pnas.1820323116), is menthol\ninhibition/binding altered in the presence of higher cation concentrations? Furthermore, a W/A\nmutation shows a gain of function phenotype, it looks as if the W/Q mutant used here also has\nthis effect, but this is not explicitly mentioned in the text. This could potentially be interesting as\nglutamine, in contrast to alanine, should retain some ion complexation ability? So does this mean that\ncation complexation at this site is not important for channel regulation?\nThank you for your comment. Regarding the complexation of ions at W583, we have performed\nthe suggested experiment, i.e. measure menthol block at a higher concentration of extracellular\nNa+. We measured block by two concentrations of menthol and observed that the blocked\nfractions are not significantly different at 140 mM and 300 mM Na+ (Fig R1). One interpretation of\nthis result is that there is no competition of menthol and Na+ for a binding site. While a density is\nindeed observed in the mentioned paper near W583, there is no evidence that this is an ion and\nthere is no experimental evidence that coordinating an ion at this position is important for any\naspect of channel functioning.\nFigure R1. Effect of extracellular Na⁺ concentration on menthol block of TRPV5. a\nRepresentative whole-cell currents recorded at –100 mV in the presence of 500 µM menthol with\n140 mM (black) or 300 mM (blue) extracellular NaCl. b Representative whole-cell currents\nrecorded at –100 mV in the presence of 3 mM menthol with 140 mM (black) or 300 mM (orange)\nextracellular NaCl. c Fractional inhibition by 500 µM (blue) and 3 mM (orange) menthol under 140\nmM and 300 mM extracellular Na⁺ conditions. Each symbol represents an individual\nmeasurement.\nWe now explicitly mention that the W583Q mutant is indeed a gain of function mutant, and we\nshow this experimentally by comparing the voltage-dependent opening of this mutant with those\nof WT, both at the single-channel and macroscopic current levels (Supplementary figure 1 and\n6). These data show that at the single channel level, W583Q opens at more positive voltages and\ngives rise to inward current also at more positive voltages than WT. Anecdotally, W583Q is highly\ntoxic to cells, something that was also described for W583A and might reflect the increased\ncurrents observed at more positive voltages.\nI wonder whether the W/Q mutant shows a similar or a more restrained GOF effect than the W/A\nmutant described in the literature. Can the authors comment on why they chose the W/Q mutant?\nWe now have performed recordings from the W583A mutant as well and show that when\ncompared to W583Q, the reduction of menthol effect and the gain-of-function phenotypes are\nsimilar (see also response to previous comment). The rationale behind the W583Q mutant is that\nintroducing a polar residue such as glutamine at this position, will abolish hydrophobic\ninteractions, while keeping a relatively bulky side chain (glutamine’s side chain volume, 143 Å3,\ntryptophan side chain volume, 228 Å3). We think that this helps us differentiate from the effect of\nresidue size from its chemical character on the interaction. We have now also analyzed other\nmutants at position W583 which strengthened the conclusion that menthol establishes specific\ninteractions with W583. See our response to your comments above.\nResults around Figure 2: It is interesting that the authors find three time constants for menthol\nconcentration-dependent closure of the channel. Can the authors speculate what these time\nconstants actually reflect on a molecular level? And is the change in the differences in tau shown\nin Figure 2E as the yellow and the blue traces actually in any way significant for the\ninhibition model the authors propose (as these seem to not significantly change with the ligand\nconcentration)?\nThank you, we admit that we did not clearly explain the results of the single\nchannel analysis and we now take this opportunity to do so. Three closed time constants can be\ninterpreted as the channel visiting at least three closed states. This is consistent with the gating\nscheme I we now use to explain the menthol data, presented in figure 1. The two fastest time\nconstants likely correspond to the transitions between closed states labelled C1 and C2 and the\nthird one, which is the slowest, corresponds to the blocked state.\nMD simulations: How different is the orientation of the Ile and Trp chains identified here for\nmenthol binding between the starting structure by Lee et al and your own cryoEM structure?\nHow mobile are these residues when menthol is present vs when it is not bound?\nIn Figure 3g, we compare the structure of TRPV5 in the presence of menthol and PIP₂ to the\nstructure with PI(4,5)P₂ alone. We find that the I575 residues do not exhibit significant positional\ndifferences, primarily because the S6 helix backbone remains unaltered. In contrast, the W583\nside chains of two subunits shift their orientation by approximately 20° to 45° to accommodate\nmenthol binding.\nTo address the reviewer’s question regarding the evolution of residue orientation, we calculated\nthe side-chain angle of isoleucine and tryptophan relative to the plane formed by the Cα atoms of\neach residue across four MD replicas (as shown in the figure R2). The periods during which\nmenthol resides in the pore are highlighted in pink. Overall, the orientation of I575 and W583 does\nnot appear to be directly correlated with the presence or absence of menthol. However, it is\ninteresting to note that in run 1, a single menthol molecule seems to induce continuous variation\nin I575 orientation (ranging from 10° to 80°), while the presence of multiple menthol molecules\n(after ~350 ns) appears to stabilize the side-chain conformation (~50°). A similar trend is observed\nin run 3, which also shows increased stabilization (~50°), likely due to multiple menthol molecules\nbeing present. Conversely, in these same runs, the orientation of W583 becomes more dynamic\nin the presence of more than one menthol molecule, fluctuating between 25° and 80°.\nFigure R2. Side-chain angle (red dash line) of isoleucine (a) and tryptophan (b) relative to the plane\nformed by the Cα atoms of each residue across four MD replicas. Pink region indicates pore occupancy\nby menthol, as showed in Supplementary Figure S9.\nWith MD you find 2 possible menthol binding sites, is there any density at all at the “upper” site in\nthe cryoEM maps? And what is the chemical nature of the contacts observed for menthol in the\ntwo binding sites in the MD simulations? Related to this, if the contacts between menthol and\nTRPV5 stem from solely from hydrophobic interactions with the Trp and Ile\nresidues, how is there any specificity for menthol at this site\nWhile our current cryoEM structure does not reveal clear density near I575 (Figure R3a, right),\nwe identified a subset of particles that display additional non-protein density near this region in a\nlocally refined reconstruction (Figure R3a, left, red circle). This map, obtained at ~3.8 Å resolution,\nsuggests the possibility of menthol binding at the \"upper\" site, albeit with greater conformational\nheterogeneity. We acknowledge, however, that this density is low-resolution and should be\ninterpreted with caution.\nIn our MD simulations, the interactions between menthol and I575 appear to be primarily\nhydrophobic in nature. Interestingly, we also observe transient hydrogen-bond interactions\nbetween menthol and W583 (0.1–0.5 ns; Figure R3b, red diamonds) as the molecule traverses\nthe pore. These interactions suggest that the donor/acceptor functional group of menthol can form\na hydrogen bond with the NH group of W583. This transient but specific hydrogen bonding may\ncontribute to selectivity beyond simple hydrophobic contacts. Therefore, although hydrophobic\ninteractions dominate, the ability of menthol to engage in polar interactions with specific residues\nsuch as W583 may help explain its preferential binding at these sites.\nFigure R3. a. Comparison of cryoEM structures shown in Figure 3 (right) and the ~3.8 Å reconstruction\nobtained from a subset of particles displaying additional non-protein density in the pore (left, red circle).\nb. Transient hydrogen-bond interactions (red diamonds) between menthol and W583 obtained in our\nMD simulation.\nDoes mutation of W/Q or I/S in silico shift the menthol occupancy to the respective “other” binding\nsite? This is a relatively straightforward experiment that would strengthen the relevance of the\ntwo binding sites (see also next point):\nWe thank the reviewer for this suggestion. Unfortunately, the computational power and time\nrequired to perform such simulations exceed the resources currently available to us, and we are\ntherefore unable to obtain sufficient sampling to unequivocally determine whether the W/Q or I/S\nmutations shift menthol occupancy to an alternative binding site. This is an important point\nbecause, even without performing in silico experiments on occupancy shifts, our\nelectrophysiological data are in agreement with the MD results showing that I575 is an important\nsite influencing menthol block, which is interesting given that it represents the narrowest part of\nthe pore and preserves, as the reviewer mentioned, the hydrophobicity of the pore. The lack of\nelectron density in this region suggests that binding is dynamic; moreover, we performed two\nmutations at this site: substitution with is polar and serine, which strongly decreased the block\neffect, and substitution with leucine, which completely preserved the block. Importantly, other\nmutants at nearby positions previously reported to affect channel gating (M578L and G579L) had\nno effect on menthol block (Supplementary figure 8), supporting the idea that I575 plays a\ntransitory role in menthol block.\nWhat was the rationale behind creating an Ile to Ser mutation? I575 is a pore constriction residue\nin TRPV5, so wouldn’t it be expected that loss of this hydrophobic residue results in loss of\nmenthol block? This may just reflect a more open pore, but does not necessarily mean this residue\nis in direct contact with menthol, something that the lack in electron density for this site also\nalludes to?\nOur rationale for introducing the I575S mutation was to test whether reducing hydrophobicity and\nside-chain volume at this pore-constriction residue alters menthol block. As the reviewer notes,\nI575 is located at the narrowest part of the TRPV5 pore, and replacing this bulky hydrophobic\nside chain with a smaller polar residue is indeed expected to affect pore geometry and\nconductance. Consistent with this, I575S reduced menthol sensitivity, an effect that can be\nexplained by changes in local pore architecture and hydrophobicity, while the absence of electron\ndensity for menthol at this site suggests that binding here may be dynamic or transient. To further\nclarify this, we also examined the I575L mutant, which preserves hydrophobicity at this position,\nand found that it retained WT-like block, whereas neighbouring substitutions (M578L and G579L)\ndid not affect block. Importantly, I575S reduced menthol sensitivity without altering overall channel\nconductance (Supplementary Fig. S6), or the sensitivity to ruthenium red block, supporting the\nidea that hydrophobic interactions at the pore constriction contribute to menthol’s blocking effect.\nTaken together, we interpret the I575S and I575L results as complementary to W583Q,\nsuggesting that residues at or near the pore constriction influence the ability of menthol to access\nand transiently occupy the conduction pathway, while W583 plays a central role in mediating the\nspecific blocking interaction.\nMinor points:\nThroughout the introduction, residue numbers are mentioned, presumably following human\nnomenclature, but possibly also rabbit as this is the channel used for this study? This needs\nspecification as the introduction discusses TRPV5 channels broadly and does not distinguish\nbetween species.\nWe thank the reviewer for this observation. Human TRPV6 contains 725 amino acids, human\nTRPV5 contains 729, and rabbit TRPV5 contains 730; however, these differences are confined\nto the C-terminus. As a result, the amino acid numbering is conserved across all three proteins.\nWe have now clarified this point in the Methods section (page 12, lines 349-351).\nFigure 2, it may be my PDF version, but the quality, especially of figure 2A, is quite poor. There\nare boxes around the plot. Maybe the authors could check that.\nThanks for pointing this out, it seems the boxes were introduced during pdf conversion, in the\ncurrent revision we have fixed these errors.\nLegend Figure 2E and F: I have stumbled a little bit over the phrasing, “the dependence of the\nthree…” (in E) and “dependence of the two time constants” (in F). It would help to really clearly\nwrite (in the main text and/or the legend) which time constants you are referring to here,\nrespectively.\nWe thank the reviewer for pointing this out. We have revised the legend (and corresponding text)\nto clearly specify which time constants are shown. In Figure 2E we now write “dependence of the\nthree closed-time constants (Table Ia)\", and in Figure 2F “dependence of the two open-time\nconstants (Table Ib).\nLine 456, spelling mistake in ‘concentration’.\nFixed\nDetail the preparation of the menthol solution in Ephys experiments, including concentration,\nsolvents used and which controls were used (it’s not clear to me whether the control was just\nbuffer or contained ethanol?).\nThanks for pointing this out. Menthol was prepared as a 200 mM stock solution in absolute\nethanol. From this solution, the adequate dilution in recording solution was prepared. These\ndilutions were then sonicated 10 min and used immediately. The control was recording solution\ncontaining ethanol at the maximum concentration used, which was 1.5 % and corresponded to\nthe ethanol content of the 3 mM menthol solution. We have now included these details in the\nMethods section (page 14, lines 421-427). Also, figures indicate the application of 1.5 % ethanol\nat the beginning of the recordings\nFor legends of figures showing Ephys data, specify that this involves TRPV5 in Hek293 cells.\nAlthough a prior structure of TRPV5 with PIP2 exists, the authors should at least briefly describe\nthe lipid binding site(s) they see in their cryoEM structure in the main text. Do menthol and PIP2\nbinding affect each other in any way?\nWe now specify in the figure legends that the recordings were obtained from rabbit TRPV5\nexpressed in HEK293 cells, and we have added a brief description of the PIP₂ binding site:\n“Consistent with this observation, we detected densities at the PI(4,5)P₂ binding site that resemble\na PI(4,5)P₂ molecule (Supplementary Figure 3e). These densities, as previously reported, are in\ncontact with the basic residues R302, R305, K484, and R584, where the interaction with R584\nbeing one of the most important for inducing the open conformational state.”\nRegarding the second question: Unlike the effect of the pore blocker Ruthenium Red or the\ninhibitor econazole, which induce conformational changes in the channel pore, mediated by\ninteractions with N574 in the former case and W495 in the latter, menthol appears to obstruct\nphysically the ion permeation pathway without inducing any evident conformational changes.\nTherefore, we conclude that menthol does not affect PI(4,5)P₂ binding.\nReviewer #3 (Remarks to the Author):\nIn the work of A. Mendez-Resendiz et al. based on data from electrophysiological experiments,\ndetermination of the spatial structure of the TRPV5 channel by cryo-EM, point mutagenesis, and\nmolecular dynamics (MD) simulations, it was concluded that menthol blocks the protein in the\nopen state. A menthol binding site formed by W583 residues has been identified in the lower gate\nof the channel. It has been suggested that W583 is involved in menthol-mediated TRPV5\ninhibition. Given the significant physiological role of this class of channels, the presence of\npathological conditions caused by disorders in their work, as well as the relatively small number\nof their known inhibitors, the topic seems relevant, and the results are necessary both for\nunderstanding the physical foundations of action of the TRP channels and for the rational design of\nnew pharmacological agents and therapies. At the same time, the interpretation of some crucial\ndata presented in the paper, a number of arguments of the authors in their favor, as well as the\nconclusions drawn, raise serious questions and doubts for me, which does not allow, in my\nopinion, to publish the work in its present form.\nThe main critical points are listed below.\nSince the methods and approaches used by the authors are currently quite routine, including in\nrelation to the study of TRPV channels, the scientific novelty of the work lies only in the\nidentification of a new inhibitor of the TRPV5 channel in the open state - menthol molecules. The\nevidence presented by the authors in favor of this hypothesis raises the following questions:\n1. The attribution of the density observed in the region of W583 in the cryo-EM model to the\nmenthol molecule is not justified. Thus, the menthol arrangement shown in Fig. 3g relative to the\nfour W583 residues does not reveal the presence of specific and strong intermolecular\ninteractions. In particular, the OH group of menthol is directed towards the nearest 6-membered\nring of indole (the distance is 3.3 A, although it is unclear between which atoms), while the methyl\ngroup of menthol forms an unfavorable contact with the NH group of the indole ring. etc. It can be\nassumed that MD results reflect the instability of this ligand arrangement - the population of such\nMD-states is significantly lower than a number of alternative states near residues I575, A576 and\nG579. At the same time, the presence of additional electron density in these areas is not observed\nin cryo-EM. This also contradicts with the phrase on page 13: “…as the I575S and W583Q-I575S\nmutations drastically reduced the potency of menthol blocking, indicating that menthol\ndynamically interacts with these two residues”. In addition, since the channel narrows in the I575\nregion, it is quite expected that menthol molecules will be located in this place, since their size\ndoes not allow for free diffusion, at least considering the pore radius (Fig. 3f) and the size of\nmenthol. But the authors do not discuss these important issues.\nWe respectfully disagree with the reviewer’s interpretation regarding the assignment of the non-\nprotein density near W583 as menthol. To evaluate whether this density corresponds to menthol,\nwe directly compared our cryo-EM reconstruction of TRPV5 in the presence of PI(4,5)P₂ and\nmenthol with a control dataset obtained under identical conditions but without menthol (Figure\nS4d). The control map, as well as other previously published TRPV5 structures in the presence\nof PI(4,5)P₂ alone, did not show any comparable non-protein density in this region. Furthermore,\nour functional data support the involvement of W583 in menthol sensitivity, providing an\northogonal line of evidence for the physiological relevance of this site.\nAs noted, our MD simulations reveal that menthol displays considerable mobility within the pore,\ntransitioning between an initial binding pose near W583 and a seemingly more stable site near\nI575. This expected dynamic behavior complicates definitive assignment of a single binding pose,\nbut reflects a potential multi-site binding mechanism, where menthol may transiently engage with\nW583 before moving deeper into the pore. The observed high mobility is consistent with menthol’s\nhydrophobic and dynamic nature.\nIt is important to mention that we have reevaluated the pose of menthol in light of the reviewer’s\nconcerns. In the refined conformation, the hydroxyl group of menthol may interact via hydrogen\nbonding with the NH group of W583, while a CH–π interaction with the indole ring of a neighboring\nW583 side chain may further stabilize its position. Whereas these contacts may not reflect\nclassical strong polar interactions, they are consistent with the types of interactions expected for\nhydrophobic ligands in similar environments. Notably, analogous CH–π interactions have been\nobserved in previous TRPV5 structures, such as in the case of econazole bound to W49512.\nImportantly, new data included in this revised manuscript, obtained from W583F and W583Y\nmutants, further support these specific interactions between menthol and W583 (Figure 4).\nAs the reviewer correctly notes, while our current cryoEM structure did not show a density near\nthe I575, MD simulations complement that result suggesting that in a dynamic system, alternative\nposes near I575, A576, and G579 may be more frequently sampled. Although we did not resolve\nhigh-resolution density in these regions, we identified a subset of particles displaying additional\nnon-protein density near I575, suggesting that this region may also accommodate menthol, albeit\nwith lower occupancy or greater conformational heterogeneity (Figure R1a, left). These findings\nare supported by functional data: the W583Q mutation partially reduces menthol sensitivity as\nwell as I575S, and W583Q/I575S double mutant shows a more pronounced effect, indicating that\nW583 may play a role in capturing menthol at the pore entrance, while I575 may contribute to\nstabilizing it in a deeper, occluding position.\nFigure R1. a. Comparison of cryoEM structures shown in Figure 3 (right) and the ~3.8 Å reconstruction\nobtained from a subset of particles displaying additional non-protein density in the pore (left, red circle).\nb. Transient hydrogen-bond interactions (red diamonds) between menthol and W583 obtained in our\nMD simulation.\n2. It is well known (see the reference Van Der Wijst (2017) and other sources, e.g., DOI:\n10.1073/pnas.1820323116) that point mutations of W583 can seriously affect the functional\ncharacteristics of the channel, therefore, the changes observed by the authors in the effect of\nmenthol on the conductivity parameters may be due to a whole set of factors, from which the role\nof menthol needs to be isolated. It seems to be a difficult task, which has not been completely\nsolved in this work. I believe that the conclusions drawn in this case are based on an overly\nsimplified picture.\nWe appreciate the reviewer’s concern regarding the separation of the gating effects of mutations\nand their effect on channel block by menthol. Our data as well as that of Van Der Wijst (2017)\nand other sources13, indeed show that there are effects of these mutations on channel gating.\nThese mutations tend to be gain of function, increasing the channel-mediated current at positive\npotentials; we have now measured current-voltage (I-V) curves for all the mutants employed in\nthis study and confirm that most of them produce a shift towards more positive potentials, which\nmeans that the channels open with less hyperpolarization (Supplementary figure 6). Given that\nmenthol is an open channel blocker, one would expect an increased occupancy of menthol and\nthus an increased blocked fraction at the same open probability. However, we observed a reduced\nefficacy of menthol, which can be better explained by mutations reducing the affinity of menthol\nto its binding site. In order to evaluate the degree that mutans affect the functionality of the\nchannel, we have measured in all the mutants the block by ruthenium red (RR) which is also a\npore blocker, whose binding site is located near the selectivity filter. Our data show that the\nmutants did not greatly affect block by RR and thus suggest that their effect on menthol is specific,\nand the result of disruptions of the binding site (Supplementary figure 7) and not drastic changes\nin the functionality of the channel.\n- It is unclear exactly how the 20 menthol molecules were positioned in the initial MD\nconfigurations - the authors only mention that “menthol molecules were placed randomly in the\naquatic region of the intracellular compartment”. However, it should be borne in mind that\nnonpolar menthol molecules can effectively interact with the protein residues closest to them,\nlocking themselves in these positions and not reaching the entrance to the channel. However,\naccording to the authors' results (Fig. S4, b), at least 10 menthol molecules were detected in the\npore, between residues I575 and W583. How is such selectivity achieved?”\nWe thank the reviewer for pointing out this oversight. The correct statement should be: “Menthol\nmolecules were placed randomly in the aqueous region.”\nIn our simulations, menthol molecules were randomly distributed in the bulk solvent on both the\nintracellular and extracellular sides of the membrane, ensuring that none were initially in direct\ncontact with the protein surface or embedded in the lipid bilayer. This setup was chosen to allow\nmenthol to explore potential binding sites spontaneously during the simulation, without biasing\nthe outcome.\nAs the reviewer correctly noted, nonpolar menthol molecules can transiently interact with nearby\nprotein residues upon initial diffusion, and indeed, we observed frequent but short-lived\ninteractions between menthol and various surface-exposed residues. However, these interactions\nwere generally unstable and did not result in persistent occupancy. For example, Figure S4d\nshows the diffusion profiles of several menthol molecules along the z-axis, where most remain in\nthe solvent phase (0–60 Å and 100–160 Å), exhibiting highly disordered motion. In a few\ninstances, such as the highlighted trajectory of one menthol molecule (light green), the ligand\nadopts a seemingly stable pose within the ARD region (~30 Å, Figure S4d) for an extended period\n(~350–500 ns).\nThe observed enrichment of menthol in the pore region, particularly between W583 and I575,\nappears to be primarily driven by hydrophobic partitioning. The inner pore of TRPV5 is notably\nhydrophobic, providing a favorable environment for menthol binding (Figure S3f). Also, using a\nrelatively high menthol concentration (~8 mM) increases the likelihood of capturing such events\nwithin the accessible simulation timescales. Nevertheless, menthol occupancy remained variable\nacross independent runs: for instance, in run 2, only two menthol molecules entered the pore,\none remaining for ~300 ns and the other for ~100 ns; in run 4, a single molecule occupied the\npore transiently for less than 200 ns.\nThese findings underscore the dynamic occupancy nature of menthol binding under our\nsimulation conditions, which aligns with its small, hydrophobic character and supports a model in\nwhich menthol acts as a fast pore blocker rather than a tightly bound ligand.\n- Parameterization of the force field for the menthol molecule was performed using an automated\nservice, without the necessary calibration on simple model systems. Since menthol is poorly\nsoluble in water, the effects of oligomerization in a polar environment are possible – these issues\nneed to be clarified in order to understand how adequate the MD results are.”\nWe thank the reviewer for raising this important point. We employed a generalized force field\nparameterization menthol using the CHARMM General Force Field (CGenFF), following an\napproach similar to that used in previous simulation studies involving menthol using another FF14.\nWhile this method is widely used in molecular simulations, we acknowledge that automated\nparameterization without further calibration against high-level quantum mechanical data or\nexperimental observables may introduce limitations.\nIn our case, the CGenFF parameterization yielded a parameter penalty score of 0.000 and a\ncharge penalty of 0.452, values that fall well below the recommended threshold for further\noptimization (penalty <10), suggesting acceptable reliability. Nevertheless, we recognize that\neven small deviations in partial charges or torsional potential can impact on the behavior of small,\ndynamic ligands like menthol in complex environments. For example, our 200-ns test simulation\nof menthol in aqueous solution indicated structural stability (RMSD ~1.5 Å), yet the computed\ndipole moment (~2.1 D) was slightly higher than previously reported computational estimates\n(~1.4 D)15. This deviation may influence menthol’s interaction profile with the pore and\nsurrounding residues, possibly contributing to the observed differences between our simulation\nresults and the cryo-EM structure.\nDespite these limitations, it is noteworthy that both our structural and simulation data consistently\nindicate that menthol occupies the intracellular entry of the pore, and that mutations at key\nresidues support a model in which it acts as a channel blocker.\nRegarding the possibility of menthol oligomerization, we did not frequently observe such behavior\nin our simulations. Figure R2 shows the lifetime of oligomerization across four replicas, with a\nmean lifetime of 0.26 ± 0.1 ns. This short oligomerization lifetime is likely because menthol\npredominantly localizes within the membrane, at the membrane–solution interface, or in contact\nFigure R2. Lifetime of oligomerization across four replicas. The mean lifetime is 0.26 ± 0.1 ns.\nwith the protein, where hydrophobic partitioning limits its self-association in bulk solvent. This\nsumulation indicates that formation of menthol complexes is short lived and not relevant for the\ninterpretation of our results.\n- When processing the experimental dose-response curves (Fig. 2 c, d; Fig. 4 d, e), the measured\nprofiles are fitted with several gaussians, although this is not justified in any way. As a result, such\na decomposition in many cases looks incorrect and, accordingly, distorts the conclusions.\nWe are sorry for the confusion. The dose responses are obtained from macroscopic current\nrecordings and are calculated as the fraction of current blocked at a given concentration of\nmenthol. These estimate does not involve fitting of gaussians. Gaussian functions were fitted to\nthe amplitude distribution of single-channel records to estimate the possible effect of menthol on\nthe current amplitude flowing through a single channel. This is necessary to distinguish between\nfast block and slow block mechanisms, as a fast blocker will reduce the current amplitude, while\na slow blocker increases the number of non-conducting events. This seems to be the case for\nmenthol as indicated by the kinetic analysis (figure 2 c-f).\n- Page 10: “The single-channel current amplitude remained unaffected by menthol, as indicated\nby all-points amplitude histograms of selected current segments (Figure 2b)”. But the histograms\nare rather different… Moreover, they are different from those reported for WT TRPV5 in (DOI:\n10.1038/srep45489) (Fig. 2f). But these effects are not discussed.\nWe thank the reviewer for this observation. In Figure 2b, the all-points amplitude histograms\nindeed differ in appearance across conditions. This variation is primarily due to changes in\nchannel open probability with increasing menthol concentrations, which alter the relative\nproportion of open versus closed events. In addition, differences in the number of events available\nat each concentration affect the shape of the histograms. Despite these changes in overall profile,\nthe position of the open-state peak relative to the closed-state peak remains constant, indicating\nthat the unitary current amplitude is unaffected by menthol.\nWe acknowledge that our histograms also differ in appearance from those reported in the cited\nstudy (DOI: 10.1038/srep45489). This discrepancy is explained by the different recording\nconditions: their single-channel currents were measured at –80 mV, whereas ours were acquired\nat –100 mV, leading to proportionally larger amplitudes under our conditions.\n- The curves in Figure S1 c, e do not reach a plateau, how in this case the Hill coefficient can be\nestimated?\nThank you for this insightful observation. We have reanalyzed the dose response curves for\nhTRPV5 and hTRPV6 channels. Specifically, the leak current was estimated and subtracted from\nthe records. We realized that this step is important specially for small amplitude currents.\nApplication of this correction yielded steeper dose response curves that can be well fitted to a Hill\nequation. We also fitted these data to a specific blocking model (Scheme I), which provides a\ngood fit to the data. Please note that this is now supplementary figure 2. The detailed derivation\nof equations used for this model is presented in a new appendix.\nIt is unclear exactly what information is needed to understand the conclusions provided by the\ngraphs of RMSF versus MD time (Figure S4, f)? The authors remarked that such plots reveal\n“minor fluctuations (~2 Å) observed at the intracellular entrance of the pore” (page 11). But what\nwas the reason to report the whole RMSF plots without discussing them?\nWe thank the reviewer for this observation. Root mean square fluctuation (RMSF) analysis\nprovides insight into the flexibility and mobility of individual residues within a molecular system\nthroughout the simulation. Regions with high RMSF values typically correspond to flexible\nsegments such as loops, termini, or intrinsically disordered regions, while low RMSF values are\nindicative of more rigid structural elements, including α-helices, β-sheets, or domains engaged in\nstable interactions. In our study, RMSF was used to evaluate whether the presence of menthol\ninduces any local destabilization or increased flexibility, particularly in the pore region of the\nchannel. As shown in Figure S4f, the average RMSF values remain relatively low (~2 Å) across\nthe transmembrane region, including the residues lining the pore and those interacting with\nPI(4,5)P . These results indicate that the pore remains structurally stable throughout the\n2\nsimulations and that menthol binding does not induce significant conformational changes in this\nregion.\n- Page 12: The meaning of the following phrase is not clear to me: “…in the absence of a\ntryptophane at position 583, menthol blocks TRPV5 by rapidly entering and exiting the conduction\npathway, albeit with lower affinity”.\nWe thank the reviewer for pointing out the lack of clarity. What we intended to convey is that, in\nthe absence of tryptophan at position 583, menthol can still interact with the pore but with reduced\nstability, resulting in rapid entry and exit from the conduction pathway and therefore a lower\napparent affinity for block. We have revised the sentence on page 6, lines 183-185 to read: “In\nthe absence of tryptophan at position 583, menthol behaves as a fast blocker, accounting for the\nremaining block.”\nReferences\n1. Schmitz, D., Shubert, V. A., Betz, T. & Schnell, M. Exploring the conformational landscape\nof menthol, menthone, and isomenthone: a microwave study. Front. Chem. 3, (2015).\n2. Linder, T., Saxena, P., Timin, E., Hering, S. & Stary-Weinzinger, A. Structural Insights into\nTrapping and Dissociation of Small Molecules in K+ Channels. J. Chem. Inf. Model. 54,\n3218–3228 (2014).\n3. Woodhull, A. M. Ionic Blockage of Sodium Channels in Nerve. The Journal of General\nPhysiology 61, 687–708 (1973).\n4. Van Der Wijst, J. et al. A Gate Hinge Controls the Epithelial Calcium Channel TRPV5. Sci\nRep 7, 45489 (2017).\n5. Hughes, T. E. T. et al. Structural insights on TRPV5 gating by endogenous modulators. Nat\nCommun 9, 4198 (2018).\n6. Lee, B.-H., De Jesús Pérez, J. J., Moiseenkova-Bell, V. & Rohacs, T. Structural basis of the\nactivation of TRPV5 channels by long-chain acyl-Coenzyme-A. Nat Commun 14, 5883\n(2023).\n7. Wang, H. & Meng, F. The permeability enhancing mechanism of menthol on skin lipids: a\nmolecular dynamics simulation study. J Mol Model 23, 279 (2017).\n8. Yang, S. et al. A Multiscale Study on the Penetration Enhancement Mechanism of Menthol\nto Osthole. J. Chem. Inf. Model. 56, 2234–2242 (2016).\n9. Hanson, S. M., Newstead, S., Swartz, K. J. & Sansom, M. S. P. Capsaicin Interaction with\nTRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation. Biophysical Journal\n108, 1425–1434 (2015).\n10. Yin, Y. et al. Visualizing structural transitions of ligand-dependent gating of the TRPM2\nchannel. Nat Commun 10, 3740 (2019).\n11. Zubcevic, L., Hsu, A. L., Borgnia, M. J. & Lee, S.-Y. Symmetry transitions during gating of\nthe TRPV2 ion channel in lipid membranes. eLife 8, e45779 (2019).\n12. De Jesús-Pérez, J. J. et al. Structural mechanism of TRPV5 inhibition by econazole.\nStructure 32, 148-156.e5 (2024).\n13. Dang, S. et al. Structural insight into TRPV5 channel function and modulation. Proc. Natl.\nAcad. Sci. U.S.A. 116, 8869–8878 (2019).\n14. Nguyen, T. H. D., Itoh, S. G., Okumura, H. & Tominaga, M. Structural basis for promiscuous\naction of monoterpenes on TRP channels. Commun Biol 4, 293 (2021).\n15. Schmitz, D., Shubert, V. A., Betz, T. & Schnell, M. Exploring the conformational landscape\nof menthol, menthone, and isomenthone: a microwave study. Front. Chem. 3, (2015)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have greatly improved the manuscript and it is now suitable for publication.", "reviewer_2": "I would like to thank the authors for their patience and the extensive comments to my previous remarks, that have been\nanswered satisfactorily. The addition of the W583F and Y mutants and the extended data and discussion of the W/A and\nW/Q mutants significantly strengthen the manuscript. While I am still not sure I fully understand the physiological relevance\nof the menthol interaction with TRPV5, the authors may have come across an interesting druggable pocket in TRPV5, but\nthis begs the question whether there are clinical indications where TRPV5 should be inhibited on purpose? It was my\nunderstanding that loss of function of TRPV5 poses a problem, i.e. in Ca resorption and renal stone formation? Do they see a\nway to leverage the menthol binding site for developing an agonist?\nSome additional minor points:\nLine 79 – TRPV5 not TRV5\nLine 80ff “Menthol also activates or inhibits other TRP channels, such as TRPV3, TRPV1 and TRPA1, 80 although the\nstructural mechanisms involved are not known (Macpherson et al. 2006; Nguyen et al. 81 2021) (Takaishi et al. 2016)\n(Kashio et al. 2024), although TRPV5 and TRPV6 are only 30 % similar 82 to other TRPVs suggesting different\nmechanisms of modulation.” Is not a complete sentence?\nLine 125 - In this case, data indicates 125 there are three closed or non-conductive states and one or two open states.\nThese kinetics are 126 consistent with the model in Scheme I.\nTechnically the data can be fitted satisfactorily with 5 states, but that doesn’t mean that there are only 5?\nLine 262 “This property makes menthol is a molecule of physiological interest.” – not a complete sentence\nLine 329 – TRPM8 not TRM8\nLine 333 – what is a “high dose exposure” of menthol? This may be mentioned explicitly to allow the reader to assess the\nrelationship between the IC50 of menthol for TRPV5 and TRPM8, and the possibility of kidney pathophysiology", "reviewer_3": "The authors have tried to address all the queries expressed in my review. It is important that the corrected version of the\narticle contains new experimental structural data obtained as a result of additional analysis. The final picture, especially in\nterms of matching the experimental and simulation data, became clearer to me.\nAt the same time, these clarifications and additions to the MD protocol and the interpretation of the simulation results are\npresent only in the reviewers' responses, but are practically not reflected in the revised version of the manuscript. In\nparticular, this is related to evaluating the reproducibility of the calculation results, the approximations made, possible\ninaccuracies in the selection of the force field, etc. Since the final conclusions of the work on the molecular mechanism of\nmenthol channel block are largely based on modeling data, I think it is important for the Reader to understand the limitations\nof the computational modeling.\nA short section could be devoted to this, for example, in the Supporting Information. This is often used in computational\nworks, which makes it possible to evaluate the predictive power of in silico models. This is especially true since there are\nsome obvious contradictions between, for example, cryo-EM and MD results – this was noticed by all the referees. In\naddition, the “Data and code availability” section does not contain information about the details of calculations - MD\nprotocols, exact force field parameters (including those for ions), etc. The availability of such data would allow the authors'\nresults to be used by other researchers in independent calculations of TRPV5 and other systems."}, "author_response": "We thank the reviewer for the assessment of our manuscript and the previous constructive comments.\nWe fully agree with the inference that loss of function of TRPV5 has clear and well documented\nconsequences in renal physiology. TRPV5 is the major apical Ca2+ entry pathway in the distal nephron, and\nreduced TRPV5 activity leads to decreased Ca2+ reabsorption, hypercalciuria, and increased risk of renal\nstone formation.\nIn this regard, identifying menthol as a TRPV5 inhibitor does not suggest any therapeutic value for menthol\nin renal disease, however, the indiscriminate use of menthol in vaping products or high-menthol exposure\nmay represent a potential health risk. This is because TRPV5 plays a key role in Ca2+ reabsorption, and its\ninhibition at elevated menthol concentrations could disturb Ca2+ handling in the kidney. For these reasons,\nTRPV5 inhibition remains biologically meaningful, even though it is not desirable as a therapeutic strategy\nfor kidney function.\nHowever, the impact of TRPV5 inhibiton may differ substantially in non-renal contexts, particularly in cancer.\nDysregulation of TRPV5/6 has also been associated with disrupted Ca²⁺ homeostasis, increased migration,\nand altered growth signaling in renal cell carcinoma and other malignancies (DOI:\n10.1016/j.juro.2011.07.086; DOI: 10.1371/journal.pone.0195844). These observations suggest that\ninhibition of TRPV5/6 may be of potential therapetic use in cancer biology.\nIn relation to the second question, defects in TRPV5-mediated Ca2+ reabsorption such as kidney stones or\nbone density reduction due to malfunctioning osteoclasts, are related to impaired plasma membrane\ntrafficking of the channel and not to channels that are unable to open\n(https://doi.org/10.1016/j.kint.2019.05.029). It thus seems like what’s needed is not a channel agonist, since\nchannel opening is not impaired but drugs that can restore trafficking to the plasma membrane. We have\nadded a paragraph with at the end of the discussion section (lines 338-342).\nAt this moment, it is not obvious to us how a channel opener could be developed from a molecule that\nblocks and stabilizes a closed conformation.\nSome additional minor points:\nLine 79 – TRPV5 not TRV5\nThanks for pointing this out, we have corrected the typo.\nLine 80ff “Menthol also activates or inhibits other TRP channels, such as TRPV3, TRPV1 and TRPA1, 80\nalthough the structural mechanisms involved are not known (Macpherson et al. 2006; Nguyen et al. 81\n2021) (Takaishi et al. 2016) (Kashio et al. 2024), although TRPV5 and TRPV6 are only 30 % similar 82 to\nother TRPVs suggesting different mechanisms of modulation.” Is not a complete sentence?\nThanks for pointing this mistake out, we have corrected the sentence.\nLine 125 - In this case, data indicates there are three closed or non-conductive states and one or two open\nstates. These kinetics are consistent with the model in Scheme I.\nTechnically the data can be fitted satisfactorily with 5 states, but that doesn’t mean that there are only 5?\nThat is correct, the number of fitted states only provides an estimate of the minimum number of states.\nMore systematic experiments and rigorous examination of alternative models would be needed to obtain a\nreliable estimate of the number of states and allowed transitions involved on TRPV5 channel gating. We\nhave added this statement in line 119.\nLine 262 “This property makes menthol is a molecule of physiological interest.” – not a complete sentence\nThanks for pointing this mistake out, we have corrected the sentence.\nLine 329 – TRPM8 not TRM8\nThanks for pointing this out, we have corrected the typo.\nLine 333 – what is a “high dose exposure” of menthol? This may be mentioned explicitly to allow the reader\nto assess the relationship between the IC50 of menthol for TRPV5 and TRPM8, and the possibility of kidney\npathophysiology\nExperimental models show that menthol produces biological effects in the hundreds of micromolar to\nmillimolar concentration range. For example, menthol causes renal proximal tubular cell toxicity at 200–\n1500 µM (DOI: 10.1124/jpet.414.129161abstract) and disrupts pulmonary epithelial and mitochondrial\nfunction in a similar range (DOI: 10.1152/ajplung.00222.2022). Toxicological work indicates that adverse\neffects appear at very high exposures, such as dietary concentrations of 24-50 mM. Although the\ncorresponding plasma menthol concentration is unknown. Together, these data indicate that TRPV5\ninhibition by menthol occurs only at concentrations that already overlap with doses known to produce\ncellular or tissue injury.\nWe have included this value in line 325.\nRegarding the TRP channel pharmacology, menthol activates TRPM8 with an EC50 of 62.6 µM (DOI:\n10.3389/fphar.2022.898670), while in our study menthol inhibited TRPV5 with an IC50 of 142.6 µM. These\nconcentrations fall in the same range where menthol has been shown to produce cellular effects, including\ntoxicity in renal proximal tubular cells and changes in epithelial and mitochondrial function. This means that,\nalthough higher than the low micromolar levels normally detected in plasma, these values are still\nphysiologically relevant. Therefore, the fact that menthol modulates TRPV5a at these concentrations\nsupports the importance of the menthol binding in TRPV5 and shows that channel modulation could occur\nunder conditions where menthol reaches elevated levels in specific tissues.\nReviewer comment:\nThe authors have tried to address all the queries expressed in my review. It is important that the corrected\nversion of the article contains new experimental structural data obtained as a result of additional analysis.\nThe final picture, especially in terms of matching the experimental and simulation data, became clearer to\nme.\nAt the same time, these clarifications and additions to the MD protocol and the interpretation of the\nsimulation results are present only in the reviewers' responses, but are practically not reflected in the\nrevised version of the manuscript. In particular, this is related to evaluating the reproducibility of the\ncalculation results, the approximations made, possible inaccuracies in the selection of the force field, etc.\nSince the final conclusions of the work on the molecular mechanism of menthol channel block are largely\nbased on modeling data, I think it is important for the Reader to understand the limitations of the\ncomputational modeling.\nA short section could be devoted to this, for example, in the Supporting Information. This is often used in\ncomputational works, which makes it possible to evaluate the predictive power of in silico models. This is\nespecially true since there are some obvious contradictions between, for example, cryo-EM and MD results\n– this was noticed by all the referees. In addition, the “Data and code availability” section does not contain\ninformation about the details of calculations - MD protocols, exact force field parameters (including those\nfor ions), etc. The availability of such data would allow the authors' results to be used by other researchers\nin independent calculations of TRPV5 and other systems.\nResponse:\nAs the reviewer suggests, we now discuss the limitations and assumptions underlying our MD simulations\nand clarify several aspects of their interpretation. We have added the following text (lines 258-271):\nAlthough our structural data indicate a stable W583-mediated menthol-binding site, the MD trajectories\nreveal additional transient interactions that show that menthol is more dynamic near the pore. Two factors\nlikely account for this behavior: the force field used (CGenFF) and the simulation temperature (29.8 °C).\nFor example, in our system the calculated dipole moment of menthol in solution is ~2.1 D, slightly higher\nthan the previously reported value of 1.4 D (ref). Likewise, the estimated menthol diffusivity at 4 °C is ~3.7\n× 10⁻¹⁰ m²/s versus ~8 × 10⁻¹⁰ m²/s at 29.8 °C. As a result, over a 100-ns trajectory menthol would, on\naverage, sample ~4.7 nm at 4 °C and ~7 nm at 29.8 °C, increasing the probability of exploring alternative\nposes in silico. Moreover, our simulations (~500 ns) are short relative to the timescales required for\nexperimental equilibration, so we cannot rule out that menthol would converge onto the single W583-\nmediated pose at longer timescales or under different sampling strategies. Despite these caveats, the MD\nresults were essential for guiding mutagenesis, and this was validated experimentally: the I575S and\nW583Q-I575S mutations drastically reduced the potency of menthol blocking, indicating that menthol\ndynamically interacts with these two residues.\nIn addition, we expanded the Methods and Supporting Information to include all details necessary for\nreproducibility, including MD protocols, force-field parameters, and relevant topology files. Specifically:\nThe topologies for menthol and PIO were generated using CGenFF server (https://cgenff.com,\nSupplementary information), then converted from CHARMM CGenFF toppar stream files to GROMACS\nformat using cgenff_charmm2gmx_py3_nx2.py (https://mackerell.umaryland.edu/charmm_ff.shtml)\ntogether with the charmm36-jul2022.ff force field (https://mackerell.umaryland.edu/charmm_ff.shtml).\nAlso, we now provide the complete menthol topology as well as all GROMACS commands used for energy\nminimization, equilibration, production runs, and analysis."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily answered my questions.", "reviewer_3": "The authors addressed all the queries expressed in my reviews. The manuscript is now suitable for publication."}, "author_response": "We thank the reviewer for constructive comments that greatly enhanced our\nmanuscript.\nWe thank the reviewer for constructive comments that greatly enhanced our\nmanuscript."}]

Decision letter