Cryo-EM structure of TRPM1 reveals a non-canonical architecture with an inverted transmembrane domain

Domain: biology
Published: 2026-03-19
Editorial outcome: minor_revision

Abstract
Transient receptor potential melastatin 1 (TRPM1) is a membrane protein essential for vision in
dim light, and mutations in TRPM1 cause complete congenital stationary night blindness.
Although TRPM1 shares sequence similarity to other TRPM ion channels such as TRPM3,
whether it independently functions as an ion channel remains controversial. This controversy is
largely caused by TRPM1’s challenging biochemical behaviors that prevent detailed molecular
characterization. In this work, we isolate TRPM1 and determine its structures using cryogenic
electron microscopy (cryo-EM). The structures reveal a canonical tetrameric fold in the
intracellular domain, consistent with other TRPM family members that are ion channels.
Surprisingly, in the transmembrane domain, despite the presence of the conserved voltage
sensor-like domain (VSLD) and pore domain (PD) in a domain-swapped fashion, the VSLD and
PD are arranged with an opposite handedness compared to other related channels. This inverted
transmembrane domain allows the formation of a large pore-like structure that supports the role
of TRPM1 as an ion channel. This non-canonical architecture of TRPM1 may also confer unique
permeation and pharmacological properties.

Introduction
In the retina, rod photoreceptors have the ability to detect single photons and transduce this
information to bipolar cells through chemical synapses 1, 2. In these bipolar cells, a signaling
cascade involving metabotropic glutamate receptor 6 (mGluR6) and transient receptor potential
melastatin 1 (TRPM1) plays a critical role in receiving light-driven signals from rods 2, 3, 4, 5, 6, 7, 8.
When there is light, TRPM1 is believed to be directly or indirectly responsible for a constitutive
depolarizing current through the bipolar cell membrane 2, 3, 4, 6, 7, 8. In the dark, however, this
TRPM1-associated current is turned off by free G proteins (Gα and Gβγ) produced by mGluR6
upon its activation (Fig. 1A) 2, 3, 4, 6, 7, 8, 9, 10. This repolarizes the bipolar cell membrane and
terminates the transduction of the vision signal 2, 3, 4, 6, 7, 8, 9, 10.

As the rod photoreceptor is mainly responsible for night vision, defects in this mGluR6-
TRPM1 pathway, such as nonsynonymous mutations in mGluR6, TRPM1 and other proteins
involved, lead to complete congenital stationary night blindness (cCSNB) 2, 11, 12, 13, 14, 15, 16, 17. So
far, more than 11 mutations in TRPM1 have been found in patients who suffer from cCSNB 2, 11,
12, 13, 14, 15, 16, 17. Some of these mutations have been shown to influence TRPM1 trafficking to the
dendritic tips in the rod bipolars cells 14, however, the majority of the TRPM1 cCSNB mutants
remain uncharacterized.

According to its amino acid sequence, TRPM1 belongs to the transient receptor potential
melastatin (TRPM) family that contains eight family members (TRPM1-8) in total 18, 19. Decades
of electrophysiological and structural analyses have unambiguously shown that the rest of the
TRPM family members (TRPM2-8) function as ion channels 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. For
example, as TRPM1’s closest homologue with ~67% sequence identity and ~76% sequence
similarity, TRPM3 has been demonstrated by patch clamp experiments to be a non-selective
cation channel in both dorsal root ganglion neurons 32 and HEK cells after heterologous
expression 22, 23. The recent cryogenic electron microscopy (cryo-EM) structures of TRPM3
reveal a canonical gate in resting and activated conformations 33, 34, 35, supporting its role as an
ion channel.

However, despite the strong sequence homology between TRPM1 and TRPM3, whether
TRPM1 itself is a bona fide ion channel is under debate 2. This debate largely stems from the
inability to reproducibly reconstitute the ion channel activity of TRPM1 after heterologous
expression in common cell lines 7, 9, 36, 37. In fact, only one study comprehensively characterized
TRPM1 current in HEK293 cells after overexpression, but the authors had to remove a large
intracellular loop or produce heteromers of TRPM1 with TRPM3 to confidently record the
TRPM1-dependent current 37. This situation is further complicated by TRPM1’s controversial
pharmacological properties 6, 8, 9, 38, 39, 40, making it difficult to correlate the results in native
bipolar cells to those after heterologous expression. For example, capsaicin was believed to
activate the endogenous TRPM1-dependent current in the bipolar cells 6, 8, 9, 38, however, some
studies also reported the presence of this capsaicin-induced current in the bipolar cells from
TRPM1 knockout mice 6, 39, and none of the studies can reconstitute the capsaicin sensitivity
with heterologously expressed TRPM1 9, 40. Other pharmacological ligands such as capsazepine
8, 9 and voriconazole 41 have also been observed to modulate TRPM1 activity. However, similar
to capsaicin, their effects were mostly tested in native bipolar cells and cannot be consistently
reproduced in heterologous systems, making it difficult to distinguish whether they result from
direct TRPM1 binding or indirect modulation by intermediate molecules. Finally, a low-
resolution negative-stain EM structure of TRPM1 reported a dimeric configuration, inconsistent
with other TRPM channels that are tetramers 42. This observation further raises the skepticism
over the function of TRPM1 as an independent ion channel.

In this work, we aim to advance our understanding of TRPM1 function on the molecular
level. To this end, we produced recombinant TRPM1 and determined its molecular structure by
cryo-EM. We also characterized its ability to permeate Ca2+ in HEK293 cells.

Results
Design and Validation of the TRPM1 Cryo-EM Construct
We chose the rat TRPM1 (rTRPM1) because the only comprehensive study that claimed to
have successfully recorded TRPM1-dependent current in HEK293 cells used the rat construct 37.
To further improve protein yield and homogeneity, we deleted the last 271 amino acids based on
its intrinsically disordered structure predicted by PSIPRED 43 and AlphaFold2 44, as well as the
sequence alignment with the TRPM3 cryo-EM construct that lacks the C-terminus 33. From now
on, we will refer to this TRPM1 construct as rTRPM1 .
EM

We first set out to determine whether the full-length rTRPM1 (rTRPM1 ) and rTRPM1
FL EM
are functional in HEK293 cells, under the hypothesis that TRPM1 functions as a bona fide ion
channel. We chose Ca2+ imaging as a readout for TRPM1 activity, as the TRPM1-dependent
bipolar cell current has been reported to be non-selective for cations such as Ca2+ 8, 38, 45, 46, 47, 48,
49. For Ca2+ imaging, we created a stable cell line using the commercially available Flp-In™ T-
REx™ 293 cell line where the expression of jGCaMP7f 50 was controlled by a doxycycline-
inducible promoter (HEK293 ). We validated the cell line with Ca2+ ionophore
doxy-jGCaMP7f
A23187, in which ~80% of the cells became fluorescent in the presence of 3 mM Ca2+
(Supplementary Fig. 1). We then transiently introduced TRPM1 constructs in HEK293 cells via
the BacMam method 51, 52. For cells transduced with both rTRPM1
FL
and rTRPM1
EM
, we
observed fluorescence increases upon Ca2+ addition among a subpopulation of cells, likely due to
the expression of TRPM1 on their surfaces (Fig. 1B and Fig. 1C). Most control cells do not
display fluorescence increase in response to Ca2+ (Fig. 1B and Fig. 1C). In the small percentage
of the control cells that showed Ca2+ permeation, their fluorescence increased by a much smaller
degree than the cells transduced with TRPM1 (Fig. 1B and Fig. 1D). These observations suggest
that the expression of both the full-length rTRPM1and the truncated rTRPM1 introduced a
EM
constitutive Ca2+ flux in HEK293 cells. This supports the role of TRPM1 as a bona fide ion
channel, which is unaffected by the large truncation introduced into our cryo-EM construct
(rTRPM1 ). A similar TRPM1-dependent Ca2+ influx in HEK293 cells has also been
EM
documented in a previous study 53.

Overall Structure of TRPM1
We first solubilized rTRPM1 in detergent glycol-diosgenin (GDN) under similar
EM
conditions as those used for the TRPM3 channel 33. However, the protein yield is poor (~80 µg
protein from 4 L HEK293 GnTI- cells) (Supplementary Fig. 2A), and subsequent cryo-EM
analyses failed to resolve the transmembrane domain (TMD) (Supplementary Fig. 3A). To
further stabilize the TMD, we incorporated 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine
(POPC), the most abundant lipid molecule on the plasma membrane, into the GDN micelles with
a detergent-to-lipid ratio of 10 to 1. The mixed micelle substantially improved the protein yield
by ~10-fold (Supplementary Fig. 2B). More importantly, the mixed micelle allowed us to
determine the structure of TRPM1 with an overall resolution of 3.6 Å with partial TMD density
resolved (Fig. 2A, Supplementary Fig. 3B and Supplementary Fig. 4). Using focused
classification and refinement, we improved the cryo-EM map of TRPM1 intracellular domain
(ICD) to 3.5 Å (Fig. 2B, Supplementary Fig. 4 and Supplementary Fig. 5A). Despite the
minimal change in the absolute value of the resolution, the map quality of ICD was substantially
improved, especially in periphery regions (Fig. 2B).

The ICD of TRPM1 displays a 4-fold symmetrical structure with two layers of the TRPM
Homology Regions (MHRs) that are very similar to other TRPM channels (Fig. 2B) 54. This
observation contrasts with an earlier negative-stain EM structure showing a dimeric structure of
TRPM1 42.

We then mapped TRPM1 mutations that cause complete congenital stationary night
blindness (cCSNB) 11, 12, 13, 14, 15, 16, 17 onto our structure (Fig. 3). These mutations are distributed
evenly across the TRPM1 structure, with 10 mutation sites in the ICD and 1 in the TMD (Fig. 3).
Interestingly, the mutation in the TMD (F1120S) has been previously reported to inhibit proper
TRPM1 trafficking in native bipolar cells 14. Notably, Fig. 3 only displays the mutations within
regions visible in our cryo-EM map. As suggested by Fig. 3, most of the mutational sites in ICD
participate in maintaining the structural integrity of TRPM1, but their precise roles need to be
evaluated by functional assays in the future.

The Unique Architecture of the TRPM1 Transmembrane Domain
In our consensus map (Fig. 2A), only the voltage sensor-like domain (VSLD) is visible in
the TMD. To resolve the architecture of the entire TMD, we set out to improve the cryo-EM map
by three-dimensional (3D) classification. Our initial hypothesis was that the rotational
heterogeneity between TMD and ICD led to poor map quality. Therefore, we first aligned the
ICD by focused refinement, then classified inside the TMD without alignment. This strategy
indeed allowed us to resolve the cryo-EM density for the pore domain (PD), but at a much lower
intensity than the VSLD (Supplementary Fig. 6). This result suggests that heterogeneity
between the VSLD and PD within the TMD also contributed to the structural dynamics.
Therefore, instead of classifying the entire TMD, we first classified the PD, then classified the
VSLD among the particles that share a similar PND (Supplementary Fig. 4). This strategy
allowed us to determine the structure of the entire TRPM1 channel to an overall resolution of 4.3
Å using C1 symmetry (Fig. 4A and Supplementary Fig. 5B). Even though the local resolution
in the TMD is around 5-6 Å (Supplementary Fig. 5B), the cryo-EM map is unambiguous for the
transmembrane helices and the connections between them (Fig. 4A and Supplementary Fig. 7).
With this map, we attempted to build a model for the TMD of TRPM1 according to its sequence
and structural similarity with TRPM3 in the VSLD and the TRP helix (Supplementary Fig. 7
and Supplementary Tab. 1).

To our surprise, TRPM1 TMD displayed a unique arrangement in which the PD from one
TRPM1 protomer contacts the VSLD of the neighboring protomer in a clockwise orientation
when viewed from the extracellular space (Fig. 5A). A similar arrangement can also be observed
in our consensus map (Fig. 2A) when viewed with low contour levels. This TMD configuration
is opposite to that of TRPM3 33, 34, 35, the closest TRPM family member to TRPM1 (Fig. 4 and
Fig. 5A-C). This TMD configuration is also opposite to those from all published structures of
ion channels with similar architectures, including all TRP channels, voltage-gated potassium
channels, voltage-gated sodium channels, and voltage-gated calcium channels 56, 57, 58, 59, 60, 61, 62,
63, 64. In other words, the TMD in TRPM1 is arranged with an inverted handedness compared to
all other related ion channels (Fig. 5A-C). Notably, the handedness here refers to the handedness
of the arrangement between VSLD and PD, not the handedness of the cryo-EM map. In fact, the
handedness of the VSLD region alone is conserved between TRPM1 and TRPM3 (Fig. 4A and
Fig. 4B).

Substantial alterations in the packing of TMD helices are necessary to achieve this inverted
configuration in TRPM1. Specifically, to reach the VSLD at the opposite side from the one in
TRPM3, the TRPM1 PD helices (S5 and S6) need to stretch out and extend at much shallower
angles with respect to the axis perpendicular to the membrane plane, resulting in a unique
curvature of S6 at its junction between the TRP helix (Fig. 4A). This possibly destabilizes S5
and S6 so that they adopt alternating conformations in neighboring protomers, making the TMD
of TRPM1 2-fold symmetrical (Fig. 5). Indeed, refinement imposing C2 symmetry yields a
better map (Supplementary Fig. 8). In one set of TRPM1 protomers, the S5 and S6 contact S1
and S4 at the extracellular side and run in parallel in the membrane (yellow and cyan in Fig. 5B
and 5C). In the other set of TRPM1 protomers, only S6 contacts S1 from the extracellular side,
while the S5 splays apart until forming close contact with S6 inside the membrane (blue and
salmon in Fig. 5B and 5C).

The Putative Ion Permeation Pathway in TRPM1
The unconventional organization of the TRPM1 TMD requires unique structural elements
to support the central ion permeation pathway. All ion channels with known structures use S6 to
form the pore. However, in rTRPM1 , S5 appears to line up the central cavity at the
EM
extracellular side (Fig. 5B, Fig. 5C, Fig. 6A and Fig. 6B). Nevertheless, at the intracellular side,
it is still S6 that forms the cavity comparable to the gate in other related channels (Fig. 5B, Fig.
5C, Fig. 6A and Fig. 6B). However, this observation does not automatically imply that the
equivalent gate residues in TRPM3 also regulate TRPM1 ion permeation. On the contrary, the
unique TMD in TRPM1 likely invokes the need for alternative gating mechanisms to control ion
permeation in TRPM1.

Globally, the inverted architecture in the TRPM1 TMD leads to an expansion of the central
cavity (Fig. 5A and Fig. 5B). Due to our limited resolution in the TMD, we cannot precisely
measure the diameter of the pore, but it is estimated to be at least ~10 Å, as the distance between
the Cα atom at the narrowest point (Y1013) is ~17.5 Å (Fig. 5D). We then compared the central
cavity between the narrowest point observed in rTRPM1 and the intracellular gate in other
EM
TRPM channels that were reported to be in the open or activated states (Fig. 5D). rTRPM1
EM
exhibited the second largest opening with a Cα-Cα distance from opposing protomers at ~17.5 Å,
about 1.5 Å narrower than the gate in the open-state TRPM2 65 (Fig. 5D). The Cα-Cα distance
between the other pair of opposing protomers in rTRPM1 is much larger due to the 2-fold
EM
symmetrical structure (Fig. 5D). On the contrary, the Cα-Cα distances at the intracellular gate in
open TRPM7 66, open TRPM5 31 and activated TRPM3 34 are 14.3 Å, 13.4 Å and 11.1 Å,
respectively. This observation is consistent with the hypothesis that TRPM1 itself is a
constitutively open ion channel 6, 8, 36, which is also supported by the results from our Ca2+
imaging experiments (Fig. 1B-D). However, we cannot rule out the possibility that some lipid
molecules can invade this pore in the cell membrane.

We then move on to test if the putative ion permeation pathway observed in our structure is
responsible for the TRPM1-dependent Ca2+ permeation observed in cells. To achieve this, we
chose the positively charged methanethiosulfonate (MTS) reagent, (2-
(trimethylammonium)ethyl) methanethiosulfonate bromide (MTSET), which is widely used in
ion channel research 67, 68, 69. The MTSET can readily conjugate to solvent-accessible cysteine
residues engineered at the channel pore and introduce a bulky side chain to prevent ion
permeation (Fig. 6C). We first confirmed that MTSET did not influence the Ca2+ permeation
ability of the wild-type (WT) rTRPM1 (Fig. 6D and Fig. 6E). We then introduced the Y1013C
EM
mutation on S5 inside the pore that is accessible from the extracellular side (Fig. 6A-C).
Notably, Y1013 forms the narrowest point in the putative pore in the rTRPM1 structure (Fig.
EM
5D). From similar Ca2+ imaging assays, this mutant was still able to permeate Ca2+ (Fig. 6D and
Fig. 6E), suggesting this cysteine mutation did not alter its function. However, MTSET
treatment substantially reduced the Ca2+ signals from this mutant (Fig. 6D and Fig. 6E).
This result suggests that the Y1013C on S5 likely directly contributes to constructing the pore.
This observation further supports the hypothesis that our cryo-EM structure of TRPM1 captures a
state that is close to its functional state in the HEK293 cell membrane.

Comparison of the ligand binding sites in TRPM3 and TRPM1
Despite the high sequence similarity in the S1-S6 helices between TRPM1 and TRPM3
(Fig. 4C), the inverted TMD architecture in TRPM1 makes it difficult to predict its
pharmacological properties entirely based on the sequence alignment. Therefore, in Fig. 7, we
compared both the sequence and the structure of the known ligand binding sites in TRPM3 and
their counterparts in TRPM1.

The VSLD of TRPM1 is structurally conserved with the VSLDs from all other TRP
channels (Fig. 4A and Fig. 4B). As a few TRPM3 ligands only interact with the VSLD, the
binding pockets for these ligands are preserved in TRPM1 (Fig. 7A and Fig. 7B). Additionally,
sequence alignment suggests that all key residues directly interacting with the ligands are also
conserved (Fig. 7A and Fig. 7B). These ligands include primidone (inhibitor) 34, 35, CIM-0216
(activator) 34, 35 (Fig. 7A), and phosphatidylinositol-4,5-bisphosphate (PIP , obligatory) 33 (Fig.
2
7B). Therefore, our TRPM1 structure suggests that all these TRPM3 ligands potentially interact
with TRPM1.

We then tested if these ligands also regulated the function of TRPM1. We chose to focus on
primidone and CIM-0216, as our Ca2+ imaging assay required the reagents to cross the cell
membrane. Consistent with the structure comparison, pre-treatment with primidone substantially
reduced the Ca2+ signal (Fig. 7C and Fig. 7D, right panels), suggesting that primidone interacted
with and inhibited TRPM1. CIM-0216, however, did not significantly change the Ca2+ signal
(Fig. 7C and Fig. 7D, left panels). The observation can be simply explained by the assumption
that the majority of TRPM1 on the cell surface adopts a wide-open pore, as in our cryo-EM
structure, that cannot be further activated. It is also possible that some population of TRPM1 on
the cell surface adopts a non-conductive state unresolved by our cryo-EM analyses. However,
the predicted binding of CIM-0216 does not induce pore opening in this population of TRPM1,
possibly because its unique TMD configuration relies on alternative gating mechanisms.

Contrary to the VSLD ligands, pregnenolone sulfate (PregS) engages at a site between the
VSLD (S1) and PD (S6 and the pore loop) in the TRPM3 channel (Fig. 7E) 35. Due to the altered
helix packing in the TRPM1 TMD that is arranged with an inverted handedness, the PregS
binding site is no longer conserved in TRPM1 despite high sequence conservation in key binding
residues — PregS will sterically clash with S6 in both types of TMD-PD interface in the 2-fold
symmetrical TRPM1 (Fig. 7E). Indeed, PregS failed to increase the TRPM1-dependent Ca2+
influx in HEK293 cells (Fig. 7F and Fig. 7G). Nevertheless, this observation can also be
explained by the already wide-open pore in TRPM1 that may not be further activated by ligands.

Methods
All plasmids and cell lines generated in the manuscript are available upon request.

Cell lines
Sf9 cells (ATCC Cat# CRL-1711) were cultured in Sf-900 II SFM medium at 27 °C.
HEK293S GnTl- cells (ATCC Cat# CRL-3022) were cultured in Freestyle 293 medium
supplemented with 2% fetal bovine serum (FBS) at 37 °C. Flp-In™ T-REx™ 293 cells (Thermo
Fisher, Cat# R78007) were cultured in DMEM supplemented with 10% FBS,1 mM L-glutamine,
100 μg/mL ZeocinTM, and 15 μg/mL blasticidin at 37 °C. HEK293 were cultured in
doxy-jGCaMP7f
DMEM supplemented with 10% FBS,1 mM L-glutamine, 100 μg/mL hygromycin and 15 μg/mL
blasticidin (DMEM B+H) at 37 °C. We did not authenticate any of the cell lines. HEK cells are
one of the commonly misidentified cell lines (https://iclac.org/databases/cross-contaminations/).

Construct design
cDNA encoding rat TRPM1 (rTRPM1) was synthesized by Twist Bioscience. Both full-
length rTRPM1 (rTRPM1 ) and rTRPM1 (rat TRPM1 ΔC2S71) were cloned into the modified
FL EM
pEG BacMam vector containing a C-terminal PreScission protease cleavage site followed by
enhanced green fluorescent protein (eGFP) 52 using NEBuilder HiFi DNA Assembly enzyme
(NEB). The Y1013C mutation (rTRPM1
EM, Y1013C
) was introduced into rTRPM1
EM
using
NEBuilder HiFi DNA Assembly enzyme (NEB).

Baculovirus generation
Bacmids carrying rTRPM1
EM
were generated by transforming DH10Bac E. coli cells with
the corresponding pEG BacMam vectors. Baculoviruses were produced by transfecting Sf9 cells
with bacmids using Cellfectin II. Baculoviruses were harvested from Sf9 cell media by filtering
the media through a 0.22 μm filter, which were then supplemented with 2% FBS for storage at
4 °C. Baculoviruses after three or four rounds of amplification were used for cell transduction.
Baculoviruses used for cell transduction were stored for no more than one month.

Ca2+ imaging
The HEK293 cell line was generated according to the manufacturer’s protocol.
doxy-jGCaMP7f
Briefly, Flp-In™ T-REx™ 293 cells were co-transfected with 2,000 ng pOG44 and 400 ng
jGCaMP7f in the pCDNA5 vector using Lipofectamine 3000. After incubation at 37 °C for 1
day, the cells were subjected to selection with 300 μg/mL hygromycin in the presence of 15
μg/mL blasticidin. Individual clonal cells were isolated using cloning cylinders and subsequently
maintained in 100 μg/mL hygromycin and 15 μg/mL blasticidin (DMEM B+H).

For Ca2+ imaging experiments, adherent HEK293 cells were grown to 80-90%
doxy-jGCaMP7f
confluency at 37 ºC in 35 mm × 10 mm tissue culture dishes in DMEM B+H. The cells were
then infected with 10% (v/v) rTRPM1 baculoviruses carrying rTRPM1 , rTRPM1 and
EM FL EM
rTRPM1 , or Sf-900 II SFM media as a vehicle control. After incubation at 37ºC for 24
EM-Y1013C
hours, the media was replaced with an expression media consisting of DMEM B+H further
supplemented with 1 µg/mL doxycycline and 5 mM sodium butyrate. The cells were incubated at
30 ºC for another 24 hours before Ca2+ imaging experiments.

Immediately prior to imaging, transduced HEK293 cells were washed twice
doxy-jGCaMP7f
with a Ca2+-free bath (1.06 mM KH PO , 2.97 mM Na HPO , 155 mM NaCl; or 10 mM Na-
2 4 2 4
HEPES pH 7.4, 145 mM NaCl, 3 mM KCl, 2 mM MgCl , 10 mM D-Glucose) to emulate
2
physiological conditions. Bright-field and GFP fluorescent images were captured using the
ECHO Revolve microscope at 20× magnification in the inverted configuration. Both the bright
field and the fluorescent images were then taken under a series of conditions as follows: 1) Basal
images were captured in the Ca2+-free bath; 2) For inhibition experiments, primidone-treated
images were captured following addition of 50 µM primidone and 1 minute incubation at room
temperature; 3) Ca2+-treated images were captured following direct addition of 3 mM Ca2+ and
gentle mixing; 4) For activation experiments, PregS-treated (Tocris 5376) and CIM-0216-treated
(Tocris 5521) images were captured following direct addition of 20 µM PregS or 10 µM CIM-
0216 after Ca2+ addition. For MTS-treated experiments with rTRPM1 , or rTRPM1 ,
EM EM, Y1013C
100 µM MTSET (Biotium 91021) was added to the cells in a Ca2+-free bath and incubated at
room temperature for 5 minutes prior to capturing the basal image. To quantify the fluorescent
images, we used an unbiased automated pipeline involving ilastik v1.4.173 and CellProfiler
v4.2.8 74. Specifically, bright-field cell images were segmented using pixel-based classification
in ilastik to generate a probability map that describes the probability of every individual pixel to
be intracellular, membrane, or background. These probability maps were then fed into the
CellProfiler program to mask individual cells in the fluorescence images. The fluorescence
intensity of every cell under different conditions was then extracted and plotted. The box plots
were generated by Qtiplot v1.2.1. The paired and independent t-tests were performed by Qtiplot
v1.2.1 with significance levels of 0.01 or 0.05.

TRPM1 expression and purification
rTRPM1
EM
was expressed in HEK293S GnTl- cells utilizing the BacMam method 51, 52.
Briefly, HEK293S GnTl- cells were grown in suspension at 37 °C until a density of ~3.5×106
cells/mL was reached, then 12% (v/v) baculovirus was added. The cells were grown at 37 °C for
another 9.5 hr, after which 10 mM sodium butyrate was added, and the growth temperature was
shifted to 30 °C. The cells were kept at 30 ° C for another 41 hr before harvest.

A POPC-GDN lipid–detergent mixture was prepared as the following. First a 1-palmitoyl-2-
oleoyl-glycero-3-phosphocholine (POPC) stock solution was prepared by drying down 10 mg
POPC in chloroform (1 mL of 10 mg/mL POPC, Avanti Polar Lipids) in a glass tube under a
gentle stream of argon. The lipid was then washed with ~2 mL pentane, followed by overnight
drying in a vacuum desiccator to eliminate residual solvent. The next day, the resulting lipid film
was hydrated with 800 µL of ultrapure water and sonicated until opalescent, yielding a
suspension of 12.5 mg/mL POPC small unilamellar vesicles. To prepare the lipid–detergent
mixed micelle, 240 µL of the POPC suspension (12.5 mg/mL) was combined with 500 µL of 6%
(w/v) GDN solution, resulting in a final 1:10 (w/w) lipid-to-detergent ratio. The mixed micelles
were incubated overnight at room temperature to ensure thorough solubilization.

Protein purification was carried out at 4 °C and was completed within 12 hr. Cell pellet
from a 4 L culture was resuspended in 200 mL buffer containing 20 mM Na-HEPES pH 7.4, 500
mM NaCl, 2 mM CaCl , 0.5 mM MgCl , 1 mM DTT supplemented with 1 mM benzamidine,
2 2
100 µg/mL Trypsin inhibitor, 5 µg/mL E-64, 11 µg/mL pepstatin A, 11 µg/mL leupeptin, 10
μg/mL aprotinin, 100 µg/mL AEBSF, and ~200 µg/mL DNase. The cell pellet was stirred until
homogeneous and gently disrupted using a Dounce homogenizer. The resulting lysate was
clarified by centrifugation at 39,000 × g for 30 min. The crude membrane pellet (~44 mL) was
resuspended with a Dounce homogenizer in 121 mL buffer containing 25 mM Na-HEPES pH
7.4, 625 mM NaCl, 2.5 mM CaCl , 0.625 mM MgCl , 1.25 mM DTT supplemented with 1.25
2 2
mM benzamidine, 125 µg/mL Trypsin inhibitor, 6.25 µg/mL E-64, 13.625 µg/mL pepstatin A,
13.625 µg/mL leupeptin, 12.5 μg/mL aprotinin, 125 µg/mL AEBSF, ~250 µg/mL DNase. The
membrane suspension was then mixed with 35 mL 6% GDN (m/v, dissolved in H O) to bring the
2
final concentration of GDN to 1.05%. The mixture was stirred for 2 hr and then the solubilized
membranes were clarified by centrifugation at 39,000 × g for 30 min. The resulting supernatant
was incubated with 3 mL GFP nanobody-coupled resin (6 mL 50% slurry) for 1 hr at 4 °C.
The resin was first washed with 90 mL wash buffer 1 containing 20 mM Na-HEPES pH 7.4,
500 mM NaCl, 150 mM KCl, 2 mM MgCl , 2 mM ATP, 1 mM DTT, and 0.00405%/0.000405%
2
GDN/POPC (m/v). The resin was then washed with 30 mL wash buffer 2 containing 20 mM Na-
HEPES pH 7.4, 500 mM NaCl, 1 mM DTT and 0.00405%/0.000405% GDN/POPC (m/v). The
resin was resuspended with 3 mL wash buffer 2, and PreScission protease was added to the resin
to a final concentration of ~0.05 mg/mL. After 1.5 h, eluted protein was collected from the flow-
through and combined with subsequent washes with 12 mL wash buffer 2. The protein was then
concentrated to ~800 µL using a 15 mL Amicon concentrator with 100 kDa molecular weight
cutoff. Concentrated protein was then clarified by centrifugation at 20,817× g for 3 min before
being further purified by size-exclusion chromatography using a Superose 6 Increase column
(10/300 GL) equilibrated with a buffer containing 20 mM Na-HEPES pH 7.4, 500 mM NaCl, 1
mM DTT, 0.00405%/0.000405% GDN/POPC (m/v). Peak fractions were pooled and
concentrated to 5.4 mg/mL using a 2 mL Amicon concentrator with 100 kDa molecular weight
cutoff.

Cryo-EM grid preparation and data acquisition
To prepare cryo-EM grids, 3.5 µL of purified rTRPM1 protein sample at 5.4 mg/mL was
EM
pipetted onto a glow-discharged Quantifoil R1.2/1.3 P400 mesh holey carbon Au grid. After 20 s
at 16 °C with a humidity of 100%, the grid was blotted for 4 s with a blotting force of 3 and flash
frozen in liquid ethane using an FEI Vitrobot Mark IV (FEI).

For cryo-EM data acquisition at the S2C2 national facility, the grid was loaded onto a 300
keV Titan Krios G3i transmission electron microscope equipped with a SelectrisX energy filter
with (10 eV slit width). The sample was collected with a Falcon 4i detector, and micrographs
were collected using EPU v3.5 software. The data collection parameters were as follows; total
micrographs: 18,322, total dose: 50 (e-/Å2), exposure time: 5.06 (s), frame time: 0.1265 (s),
physical pixel size: 0.954 (Å), dose per frame: 1.25 (e-/ Å2/frame), dose rate: 8.99 (e-/pixel/s),
and a nominal defocus range of -0.6 to -1.8 (μm).

Cryo-EM image processing and model building
Cryo-EM movie stacks were gain-normalized and corrected for beam-induced motion
using CryoSPARC v4.7.1 75 Patch Motion Correction. Parameters for the contrast transfer
function (CTF) were estimated from motion-corrected summed images without dose-weighting
using CryoSPARC v4.7.1 Patch CTF Estimation.

All subsequent cryo-EM image processing was performed on dose-weighted motion-
corrected summed images. The initial data processing was done in CryoSPARC v4.7.1. Briefly,
particles were first picked by the automatic Blob Picker and extracted, which were then sorted by
several rounds of 2D classification. The selected particles after 2D classification were subjected
to ab-initio reconstruction in C1. The resulting reconstructions were subsequently used as models
for heterogeneous refinement to recover some good particles discarded by 2D classification.
Finally, the good particles sorted by heterogenous refinement were moved forward with further
3D classification in RELION v5.0-beta-0-commit-832d07 76, 77. These particles were first sorted
by a global classification, and the resulting particles that gave reconstructions with channel
features were pooled and refined first without a mask, then with a mask covering only the
intracellular domain (ICD). To increase the resolution in the ICD, particles were symmetry-
expanded in C4 and were then subsequently refined with a mask covering the ICD. This is
roughly equivalent to refinement with C4 symmetry. To resolve the transmembrane domain
(TMD), the non-expanded particles were classified without alignment, focused on a mask
covering the pore domain (PD). The particles that displayed the most continuous pore density
were then pooled and further classified without alignment using a mask covering only the
voltage sensor-like domain (VSLD). The final particles were further refined in CryoSPARC
v4.7.1 in C1 or C2 locally using the mask covering the entire TRPM1.For all cryo-EM maps, the
mask-corrected FSC curves were calculated in cryoSPARC v4.7.1 and the resolutions were
reported based on the 0.143 criterion 78.

The initial model was obtained by docking the TRPM3 model (PDB ID: 8ED7)
[https://www.rcsb.org/structure/8ED7] into the cryo-EM map of TRPM1. The sequence was then
edited, and the coordinates were manually adjusted to fit the cryo-EM map using Coot v0.9.8 79.
Particularly, the TMD was modified by cutting out individual S5 and S6 helices in TRPM3 and
rotating them independently to fit the densities in the TRPM1 map. To facilitate model building
with an intermediate-resolution cryo-EM map, EMReady v1.2.1 80 was utilized to enhance map
quality, especially in regions exhibiting weak density. EMReady was able to substantially
improve the map in the ICD region, however, only minimal improvement was observed in the
TMD region. Therefore, we utilized the EMReady-enhanced map generated from C4 symmetry-
expanded particles to aid model-building in the ICD. On the other hand, to build the TMD
helices, we used the map refined in either C1 or C2, as the C4 symmetry is broken in the TMD.
Due to the limited resolution in the TMD, we used ISOLDE v1.7.1 81 to improve geometry and
model-to-map correlation. The models were then iteratively edited and refined in Coot v0.9.8,
ISOLDE v1.7.1 , and PHENIX v1.21.2-5419 82. The quality of the final models was evaluated by
MolProbity 83 in PHENIX v1.21.2-5419 . All figures were prepared using UCSF ChimeraX v1.7
84.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This elegant study by Fabrizio et al. resolves several outstanding controversies regarding the structure and function of\nTRPM1, the transduction channel in retinal on-bipolar cells. The authors demonstrate calcium influx through their truncated\nTRPM1 channel expressed in HEK cells, strongly indicating that TRPM1 is a constitutively open cation channel. Their cryo-\nEM results reveal the expected structural similarity to other TRPM1 channels, especially TRPM3, with an intriguing\ndifference in the transmembrane domain, where the relative orientations of the VSLD and PD are opposite in TRPM1\ncompared to all other related ion channels. This results in a large pore that is predicted to confer unique conduction\nproperties on TRPM1.\nThe authors removed the C-terminal 271 amino acids to aid the cryo-EM; however, it is important to determine whether full-\nlength TRPM1 is also constitutively permeable to calcium when expressed in HEK cells.\nThe authors write that there are “no known potent activators or inhibitors that selectively target TRPM1” (Intro, pg 4, ln 53).\nThe authors neglect to reference a paper by Xiong et al., 2015 showing that voriconazole is a selective inhibitor of TRPM3\nand also blocks the ON-bipolar cell light response. It would be a simple experiment to show whether voriconazole blocks\ncalcium influx through rTRPM1-EM or FL-TRPM1 in HEK cells. Similarly, it would be worth testing primidone, a TRPM3\ninhibitor, as the binding site is conserved in TRPM1. These experiments would increase the impact of the study.\nMinor comments:\nIntro, pg 3, ln 40: Percent sequence identity and similarity appear to be reversed. There should be greater sequence\nsimilarity than identity.\ncCSNB should be mentioned in the Introduction to provide some context and relevance for Figure 3.\nResults, p7, Ln 130-131 “Similar arrangement can also be observed in our consensus map (Fig. 3A) when viewed with low\ncontour levels.” Shouldn’t this sentence refer to Fig. 4A, rather than 3A?\nDiscussion, Pg.10, Ln 205-206: “Our structural and functional data are also consistent in the inability of PregS to modulate\nTRPM1 Ca2+ permeability (Fig. 1B, Fig. 1C, and Fig. 5A).” This sentence should refer to Figs. 1B, 1C, and 6C, not 5A.", "reviewer_2": "This manuscript reports cryo-EM structural studies of TRPM1. In contrast to other TRP(M) channels, the transmembrane\ndomain of TRPM1 adopts a unique arrangement between the voltage-sensor and pore domains, which generates a large,\ncentral pore. This new structure, if physiologically relevant, provides important insights into the diversity of TRP channel\nstructure and function. Overall, the structural analysis provides interesting new features of TRP channels. However,\nfunctional analysis and physiological relevance of the reported TRPM1 structure are not well addressed. Below are some\nspecific comments.\nMajor points:\n1. The authors stated that the TMD of TRPM1 has an overall resolution of 3.6 angstroms. However, the cryo-EM densities in\nFigures 2 and 4 and in supplementary figures do not appear to be of such high quality. It is important to show cryo-EM\ndensities for each TM helix and the fit of atomic models to better evaluate the quality of the cryo-EM map. This is critical for\ninterpretation of the channel structure.\n2. A table summarizing cryo-EM data acquisition and structural refinement statistics is not provided. PDB validation report is\nalso not provided.\n3. As shown in Fig. 5A, the unique VSD-PD arrangement of TRPM1 appears to result in a very large pore in the center. The\nauthors claimed that the diameter of the pore cannot be measured but it is estimated to be at least 10 angstroms. Are side\nchains considered in this estimation? Again, it is important to show the cryo-EM densities of the pore domain and the fit of\nthe transmembrane helices. The pore diameter estimation should also be more rigorously done. Even with limited resolution,\nfor instance, calculation of the diagonal distances between the backbone C-alpha atoms and comparison with closely\nrelated TRPM channels would be informative.\n4. The authors suggest that the large pore observed in TRPM1 explains the constitutively open nature of the channel. Would\nit be possible to introduce point mutations of the pore-lining residues to close the channel pore and test these mutations\nusing calcium imaging experiments? It seems that electrophysiological recordings of TRPM1 have not been achieved.\nMinimally, the physiological relevance of the structure should be tested with calcium imaging experiments. These\nexperiments would further support the authors’ conclusion that TRPM1 is an ion channel.\n5. As shown in Figure 6A and 6B, the binding sites for some TRPM channel ligands are conserved in TRPM1. It would be\nimportant to functionally test whether these ligands have any effect on TRPM1 channel function.\n6. Figure 5 panel C, the diagram indicates that S5 helices are lining the central ion pore. Fig. 5A also shows that S5\ncontributes to the construction of the ion pore. Please add a figure to show how the central ion pore is constructed.\nMinor points:\n1. Page 3 Line 40, please correct the numbers for sequence identify and similarity. It states 76% identify and 67% similarity.\n2. Please provide orientation plots of the particles used for final reconstructions."}, "author_response": "Response to the Reviewers\nFirst, we would like to thank both reviewers for the thoughtful and constructive comments that\nhave substantially improved the manuscript.\nReviewer #1 (Remarks to the Author):\nThis elegant study by Fabrizio et al. resolves several outstanding controversies regarding the\nstructure and function of TRPM1, the transduction channel in retinal on-bipolar cells. The authors\ndemonstrate calcium influx through their truncated TRPM1 channel expressed in HEK cells,\nstrongly indicating that TRPM1 is a constitutively open cation channel. Their cryo-EM results\nreveal the expected structural similarity to other TRPM1 channels, especially TRPM3, with an\nintriguing difference in the transmembrane domain, where the relative orientations of the VSLD\nand PD are opposite in TRPM1 compared to all other related ion channels. This results in a large\npore that is predicted to confer unique conduction properties on TRPM1.\nThe authors removed the C-terminal 271 amino acids to aid the cryo-EM; however, it is important\nto determine whether full-length TRPM1 is also constitutively permeable to calcium when\nexpressed in HEK cells.\nWe performed the same Ca2+ imaging assay with HEK293 cells expressing full-length rat TRPM1,\nwhich unambiguously showed that the full-length construct is also able to support Ca2+ permeation.\nWe have now included this result in Fig. 1B-D and described it in lines 92-99, in the first paragraph\nunder “Design and Validation of the TRPM1 Cryo-EM Construct”.\nThe authors write that there are “no known potent activators or inhibitors that selectively target\nTRPM1” (Intro, pg 4, ln 53). The authors neglect to reference a paper by Xiong et al., 2015\nshowing that voriconazole is a selective inhibitor of TRPM3 and also blocks the ON-bipolar cell\nlight response. It would be a simple experiment to show whether voriconazole blocks calcium influx\nthrough rTRPM1-EM or FL-TRPM1 in HEK cells. Similarly, it would be worth testing primidone,\na TRPM3 inhibitor, as the binding site is conserved in TRPM1. These experiments would increase\nthe impact of the study.\nOur initial intention was to keep the introduction on pharmacological regulation as simple as\npossible as the results are largely controversial. In addition, most of the ligands, if not all, have not\nbeen shown to act upon reconstituted TRPM1, making it difficult to distinguish if they directly\nbind and gate TRPM1, or if they modulate TRPM1 via intermediate molecules in the retinal bipolar\ncells. However, we do agree that it is better to include more information on this subject to\nfamiliarize the readers with the field. Therefore, we added lines 56-69 in the introduction to expand\non previous findings related to pharmacological regulators.\nWe pre-incubated cells expressing TRPM1 with 100 μM voriconazole and did not observe a\nsignificant decrease in the Ca2+ signal. We think voriconazole might be similar to capsaicin, which\nis able to elicit a strong effect in retinal bipolar cells, but the effect might be related to some\nintermediate components involved in TRPM1 gating. Consistent with this hypothesis, in Xiong et\nal. 2015, voriconazole almost completely inhibited the TRPM1 current (~90%) elicited by\ncapsaicin, but only partially (~75%) inhibited the TRPM1 current elicited by mGluR6 antagonism.\nWe also acknowledge that voriconazole inhibits TRPM3 in a reconstituted setting (CHO and\nHEK293 cells, Xiong et al. 2015), however, as the transmembrane domains of TRPM1 and\nTRPM3 are very different, it is possible that the binding site for voriconazole on TRPM3 is no\nlonger conserved for TPRM1, or that the gating mechanism between TRPM1 and TRPM3 upon\nvoriconazole binding is different. Finally, we want to emphasize that even though we demonstrated\nTRPM1 to work independently as an ion channel in this manuscript, we strongly believe that the\nendogenous environment in the retinal bipolar cells, including the local lipid environment and\nprotein partners, are essential for reconstituting the native pharmacological properties of TRPM1.\nAs this result is not directly related to our central aim to report the first structure of TRPM1, we\ndecided not to include it in this manuscript. However, we plan to systematically characterize all\nreported pharmacological regulators on TRPM1 using our Ca2+ imaging assay and report the\nresults in an independent manuscript that will be developed in the future.\nWe pre-incubated cells expressing TRPM1 with 50 μM primidone and observed a significant\ndecrease in the Ca2+ signal (Fig. 7C-D and Lines 227-231). This is consistent with the prediction\nbased on our cryo-EM structures. This result also suggests that our Ca2+ imaging-based assay is\nable to pick up inhibition, indicating the HEK293 cells may lack certain components to support\nthe voriconazole sensitivity of TRPM1.\nMinor comments:\nIntro, pg 3, ln 40: Percent sequence identity and similarity appear to be reversed. There should be\ngreater sequence similarity than identity.\nWe have corrected the numbers.\ncCSNB should be mentioned in the Introduction to provide some context and relevance for Figure\n3.\nWe have now added a paragraph in the introduction (currently the second paragraph) to provide\nsome context for the cCSNB mutations.\nResults, p7, Ln 130-131 “Similar arrangement can also be observed in our consensus map (Fig.\n3A) when viewed with low contour levels.” Shouldn’t this sentence refer to Fig. 4A, rather than\n3A?\nWe have now corrected this mistake.\nDiscussion, Pg.10, Ln 205-206: “Our structural and functional data are also consistent in the\ninability of PregS to modulate TRPM1 Ca2+ permeability (Fig. 1B, Fig. 1C, and Fig. 5A).” This\nsentence should refer to Figs. 1B, 1C, and 6C, not 5A.\nWe have now moved this figure to Fig. 7 and corrected this mistake.\nReviewer #2 (Remarks to the Author):\nThis manuscript reports cryo-EM structural studies of TRPM1. In contrast to other TRP(M)\nchannels, the transmembrane domain of TRPM1 adopts a unique arrangement between the\nvoltage-sensor and pore domains, which generates a large, central pore. This new structure, if\nphysiologically relevant, provides important insights into the diversity of TRP channel structure\nand function. Overall, the structural analysis provides interesting new features of TRP channels.\nHowever, functional analysis and physiological relevance of the reported TRPM1 structure are not\nwell addressed. Below are some specific comments.\nMajor points:\n1. The authors stated that the TMD of TRPM1 has an overall resolution of 3.6 angstroms. However,\nthe cryo-EM densities in Figures 2 and 4 and in supplementary figures do not appear to be of such\nh of such\nhigh quality. It is important to show cryo-EM densities for each TM helix and the fit of atomic\nmodels to better evaluate the quality of the cryo-EM map. This is critical for interpretation of the\nchannel structure.\nWe agree that our original wording is confusing. We meant to say we were able to resolve the\nentire protein to an overall resolution of 3.6 Å, and in this 3.6 Å consensus map we could also see\nsome features in the transmembrane domain. In the current draft, we reworded this sentence to\n“More importantly, the mixed micelle allowed us to determine the structure of TRPM1 with an\noverall resolution of 3.6 Å with partial TMD density resolved” in lines 111-113. We additionally\nstated the local resolution of the TMD in lines 142-143, and added the cryo-EM density for each\nTM helix in the new Supplementary Fig. 7. We acknowledge the low resolution in the\ntransmembrane domain, but we are confident about our claim with the altered organization based\non the clear connectivity between S4 and S5. We are also confident about the constitutive open\npore — even though we cannot see sidechain densities, the opening is too wide to be closed.\n2. A table summarizing cryo-EM data acquisition and structural refinement statistics is not\nprovided. PDB validation report is also not provided.\nWe have now provided a supplementary table 1 and attached the PDB validation report to this re-\nsubmission.\n3. As shown in Fig. 5A, the unique VSD-PD arrangement of TRPM1 appears to result in a very\nlarge pore in the center. The authors claimed that the diameter of the pore cannot be measured but\nit is estimated to be at least 10 angstroms. Are side chains considered in this estimation? Again, it\nis important to show the cryo-EM densities of the pore domain and the fit of the transmembrane\nhelices. The pore diameter estimation should also be more rigorously done. Even with limited\nresolution, for instance, calculation of the diagonal distances between the backbone C-alpha\natoms and comparison with closely related TRPM channels would be informative.\nWe previously stated at least 10 angstroms based on the diagonal distance between backbone Cα\nmeasurement of ~17.5 Å. We have now more rigorously presented this data and also compared\nTRPM1 with other TRPM channels that are claimed to be in the open or activated conformation\nin Fig. 5D and Lines 186-194. As TRPM1 may not have a canonical gate due to its unique\ntransmembrane domain, in Fig. 5D, we picked the narrowest point in our TRPM1 structure and\ncompared it to the distance at the canonical intracellular gate residues in all other TRPM structures.\nWe have now included Supplementary Fig. 7 to show the fit of all transmembrane helices, which\nincludes the pore domain S5 and S6.\n4. The authors suggest that the large pore observed in TRPM1 explains the constitutively open\nnature of the channel. Would it be possible to introduce point mutations of the pore-lining residues\nto close the channel pore and test these mutations using calcium imaging experiments? It seems\nthat electrophysiological recordings of TRPM1 have not been achieved. Minimally, the\nphysiological relevance of the structure should be tested with calcium imaging experiments. These\nexperiments would further support the authors’ conclusion that TRPM1 is an ion channel.\nAs the pore of TPRM1 is quite large, we are concerned that even a lysine mutation might not be\nable to block ion permeation. Additionally, we will need to normalize the surface expression levels\nfor the mutants and the WT, which is technically challenging given the low expression level of\nTRPM1 on the cell surface.\nTherefore, instead, we took the approach with the traditional MTS reagent. We chose the\ntryptophan residue on S5 (Y1013) located in the membrane, accessible from the extracellular side\nand predicted to be pore-facing in all protomers (Fig. 6A-B). This Y1013 is also the narrowest\npoint in the pore of TRPM1 (Fig. 5D). We mutated this Y1013 to cysteine for conjugation with the\nMTS reagent. Using our Ca2+ imaging-based assay, we demonstrated that MTS treatment of this\ncysteine mutant substantially reduced the Ca2+ signal; however, the cysteine mutant without MTS\ntreatment, or WT TRPM1 with MTS treatment, was still able to support Ca2+ permeation (Fig. 6D-\nE).\nThis result provided a connection between the cryo-EM structure and physiology of TRPM1 in the\ncell membrane and substantially strengthened our manuscript. We completely rearranged the text\nand added a new section titled “The Putative Ion Permeation Pathway in TRPM1” to accommodate\nthis new result.\n5. As shown in Figure 6A and 6B, the binding sites for some TRPM channel ligands are conserved\nin TRPM1. It would be important to functionally test whether these ligands have any effect on\nTRPM1 channel function.\nThese experiments have also been suggested by the first reviewer. We tested the effect from\nprimidone and showed that it substantially inhibited the TRPM1-dependent permeation (Fig. 7C-\nD). We also tested the effect from CIM-0216, however, it did not alter the Ca2+ signal (Fig. 7C-D).\nWe raised two hypotheses, one is that most TRPM1 on the cell surface adopt the fully open\nstructure as what we see in cryo-EM, and we cannot further open these channels by any agonist.\nAnother hypothesis is that there is some population of closed TRPM1, however, the binding of\nCIM-0216 is not sufficient to open the pore given the unique transmembrane domain in TRPM1.\nThese results are discussed in Lines 227-237. We did not test the effect of PIP , as directly adding\n2\ndisruption, insufficient distribution in the inner leaflet, and the presence of PIP in the outer leaflet.\n2\ntranslocation, inside-out electrophysiology and other methods. However, in this manuscript, we\nhope to focus on reporting the structure of TRPM1. We will perform those experiments in the\nfuture and develop another manuscript that entirely focuses on PIP regulation.\n2\n6. Figure 5 panel C, the diagram indicates that S5 helices are lining the central ion pore. Fig. 5A\nalso shows that S5 contributes to the construction of the ion pore. Please add a figure to show how\nthe central ion pore is constructed.\nThe new figure (Fig. 6) added to describe the results from MTS conjugation also showed how the\npore is constructed from both the top view (Fig. 6A) and the side views (Fig. 6B).\nMinor points:\n1. Page 3 Line 40, please correct the numbers for sequence identify and similarity. It states 76%\nidentify and 67% similarity.\nThis has also been mentioned by the first reviewer and we have now corrected this mistake.\n2. Please provide orientation plots of the particles used for final reconstructions.\nWe have now included the angular distribution of our particles in Supplementary Fig. 5."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns in the original submission. I have a few minor suggestions to improve the resubmission:\nLines 224: Please indicate that primidone is an inhibitor and CIM-0216 an activator of TRPM3. This will make the reasoning in the following paragraph clearer to the reader.\nThe following sentence (lines 234-237) is confusing; please rewrite, preferably as two sentences:\n“It is also possible that some population of TRPM1 on the cell surface adopts a non-conductive state unresolved by our cryo-\nEM analyses, however, the predicted binding of CIM-0216 cannot induce the pore opening due to the altered TMD\norganization in TRPM1 compared to TRPM3.”\nLines 150-151: “A Similar arrangement can also be observed in our consensus map (Fig. 2A) when viewed with low contour levels.” Shouldn’t this sentence refer to Fig. 4A, rather than 2A? Also, the \"s\" in similar should not be capitalized.", "reviewer_2": "The authors have adequately addressed my concerns, and the manuscript is now suitable for publication."}, "author_response": "Response to the Reviewers\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all my concerns in the original submission. I have a few minor\nsuggestions to improve the resubmission:\nLines 224: Please indicate that primidone is an inhibitor and CIM-0216 an activator of TRPM3.\nThis will make the reasoning in the following paragraph clearer to the reader.\nThis sentence has now been reworded to “These ligands include primidone (inhibitor), CIM-0216\n(activator) (Fig. 7A), and phosphatidylinositol-4,5-bisphosphate (PIP , obligatory) (Fig. 7B).”\n“It is also possible that some population of TRPM1 on the cell surface adopts a non-conductive\nstate unresolved by our cryo-EM analyses, however, the predicted binding of CIM-0216 cannot\ninduce the pore opening due to the altered TMD organization in TRPM1 compared to TRPM3.”\nThis sentence has now been reworded to “It is also possible that some population of TRPM1 on\nthe cell surface adopts a non-conductive state unresolved by our cryo-EM analyses. However, the\npredicted binding of CIM-0216 does not induce pore opening in this population of TRPM1,\npossibly because its unique TMD configuration relies on alternative gating mechanisms.”\nLines 150-151: “A Similar arrangement can also be observed in our consensus map (Fig. 2A)\nwhen viewed with low contour levels.” Shouldn’t this sentence refer to Fig. 4A, rather than 2A?\nAlso, the \"s\" in similar should not be capitalized.\nThe “s” in “similar” has now been changed to lowercase. Regarding the figure, we are actually\nreferring to Fig. 2A here to show that the arrangement in the transmembrane domain that we sorted\nout after 3D classification (Fig. 4A) is similar to the ones before classification (Fig. 2A).\nReviewer #2 (Remarks to the Author):\nThe authors have adequately addressed my concerns, and the manuscript is now suitable for\npublication."}]

Decision letter