Ion Channels:

Application Note

TMEM175,

High throughput organellar
electrophysiology of TMEM175 and TPC2
from freshly isolated lysosomes recordedon the SyncroPatch 384

TPC2Cells:
HEK-293,
lysosomes
Tools:
SyncroPatch 384

Axxam S.p.A., Milan.
Nanion Technologies GmbH, Munich.

Summary
Intracellular ion channels are known to play an essentialof low quantity (cardiomyocytes, iPS cells or organelles) - torole in various signaling pathways for health and disease,

reduce costs and save time.

considering that over 80% of transport processes occur inside
the cells 1. Among the variety of organellar channels andOur approaches resulted in the construction of cumulativetransporters the proton leak channel transmembrane protein

concentration response curves and even intraluminal solution

175 (TMEM175) and the lysosomal two-pore channel (TPC)

exchange during the recording from freshly isolated lysosomes

have received increasing attention in the field given theirhighlighting the broad range of applications possible with thepotential roles in connecting lysosomal homeostasis with

SyncroPatch 384.

pathophysiological conditions such as Parkinson’s disease andcancer 2-4. Consequently, the interest to explore intracellular

Results

ion channels as therapeutic targets has grown tremendously
indicating a need for high-throughput electrophysiology

TMEM175

including patch clamp. There has been some progress inalternative approaches such as solid supported membrane

Enlarged lysosomes incubated with 1 µM Vacuolin-1 wereelectrophysiology (SSME using the SURFE2R 96SE) recently 5,

stained with 0.1 µM LysoTracker™ Red DND-99 (Invitrogen), ahowever, until now, HTS patch clamp has lacked the possibility

red fluorescent dye that stains acidic cellular compartments,

to collect data from native lysosomes.

such as lysosomes. The dye was added to the cells before
isolation of the lysosomes and images were acquired at

Axxam and Nanion Technologies have now developed assays

different magnifications and dilutions using the Operetta

to investigate the function and pharmacology of lysosomal

system (Perkin Elmer), resulting in an average diameter ofchannels under native conditions, providing groundbreaking

2.1 µm (Figure 1).

tools for the drug discovery industry. This is possible due to thedevelopment of special consumables (single- and multi-hole)dedicated to pursuing organellar recordings in combination
with the high flexibility of the SyncroPatch 384 utilizing an ultralow cell density approach that can use as low as 50k cells/ml,
and small volumes of 1 ml for the whole of the 384-well plate,
without a drastic reduction in success rate. This can be ofA

B
8Diameter (µm)

extreme importance for expensive - as well as for samples

Figure 1: A Isolated lysosomes stained withLysoTracker™ Red DND-99 (Invitrogen); images
at different magnifications were acquired usingthe Operetta (Perkin Elmer). B Average diameter
calculated at different dilutions: 3.0 ± 2.1 µm (1:10);
2.0 ± 1.3 µm (1:20); 2.4 ± 1.7 µm (1:50); data arepresented as mean ± SD.

6420
0
1:1

nio

lut

di0
1:2

nio

lut

di0
1:5

niolutdiThe remaining lysosomes were used on the SyncroPatch 384

4-AP, acidification of the luminal solution, due to the internal

for recording TMEM175 channels expressed endogenously inexchange from pHluminal 7.0 to 4.0, increases TMEM175 currentHEK-293 cells.

(Figure 3 B).

Critical for success was usage of Nanion’s “Organellar Chips”,

A similar experiment was repeated by inverting the luminala specialized consumable that supported and maintained

pH, starting from 4.0 and changing to 7.0, using the internal

the integrity of lysosomes throughout the recording andperfusion feature of the SyncroPatch 384. In the presence

supported cumulative concentration response curves ofof 4-AP, the reduction of H+ in the luminal solution induces

DCPIB, a novel TMEM175 activator, able to mediate H and K

a reduction in TMEM175 current due to a lower proton

currents 6 as highlighted in Figure 2. Since TMEM175 channels

contribution (Figure 3 C-D).

++

release luminal H+ into the cytosol, we developed assays using
luminal solutions with different pH values, to enhance proton
conductance, in addition to potassium flux. The seal resistancein “whole-lysosome” configuration was calculated before
compound application and shows average values of 1.4 ±0.2 GΩ and 2.1 ± 0.6 GΩ at pHluminal 4.0 and 7.0, respectively.
TMEM175 activation was accompanied by a drop in Rseal,indicative for stimulation of a leak channel (Figure 2 A).

TPC2Enlarged lysosomes (Vacuolin, 1 µM) were freshly isolated
as described in Schieder et al 8,9 from HEK cells either stably
expressing TPC2-YFP 10-12 or without overexpression to record
endogenous lysosomal currents using Nanion’s “OrganellarChips”. Lysosomes contained high amounts of TPC2 andWe then executed cumulative concentration additions ofDCPIB to activate endogenous TMEM175 channels using only
part of the NPC-384 chip (32 wells per condition). Our analysis
reveals an EC50 of 65.3 ± 17.5 µM (n=5) at pHluminal 4.0 and 21.5
± 4.1 µM (n=3) at pHluminal 7.0 for outward currents (ion andproton flux from lumen to cytosol), as shown in Figure 2 D.

Representative traces (Figure 2 B-C) clearly show a larger
TMEM175 current evoked in the presence of the highest DCPIB
concentration in an acidic luminal environment, suggestingenhanced proton flux at acidic luminal pH.

revealed diameters in the range of few micrometers asvisualized in Figure 4 A, similar to what was seen during therecordings of endogenous TMEM175 (Figure 1). Based on
the epifluorescence, we could also determine a densityof 100k lysosomes per ml that was sufficient to execute ourstudies. For TPC2, we employed the ultra-low cell density
approach, where we observed lysosomal seals in ‘whole-cell’
mode that remained stable with >1 GΩ seals throughout therecording (Figure 4 B). Subsequently, we were able to elicit
lysosomal currents by addition of the specific TPC2-activator
TPC2_A1_P 13,14 that was absent when only reference solution
was applied (Figure 4 C-D). As anticipated, the activation wasGiven the known pH dependence of TMEM175 activity 7
we also employed intraluminal solution exchange for thefirst time where we observed a current modulation after
changes in luminal pH. During the experiment with pHluminal

7.0, TMEM175 current was first evoked by DCPIB application,then partially blocked by 4-AP (Figure 3 A). In the presence ofaccompanied by a drop in Rseal, which was not observed
when reference solution was applied, indicative for stimulationof a (cation) leak channel (Figure 4 B-D). Furthermore, we
performed cumulative concentration response curves on thelysosomes using TPC2_A1_P in experiments lasting ~ 15 minutes.
Our analysis reveals an EC50 of 5.4 ± 1.6 µM (n = 32) for inward
currents measured at -100 mV and 6.6 ± 2.7 µM (n = 32) for
outward currents at +80 mV in good agreement with theliterature 13,14 as highlighted in Figure 5.

A

BR seal(M Ω )

3000

800

DCPIB 100 µM
DCPIB 50 µM
DCPIB 25 µM
DCPIB 12.5 µM
vehicleI TMEM175 (pA)

600
2000

400

100001010CPID

DCPIB 100 µM
DCPIB 50 µM
DCPIB 25 µM
DCPIB 12.5 µM
vehicleI TMEM175 (pA)

600
400200mV-100-50-200-400-200

K+ /H+
pH 4

100

D600

800

pH 7Na+ /K+50-40000µM

pH-50BB
PIDCC-100

7.
0

4.
pHµM

pHcle

hihi
ve

cle

pH

7.

4.

000ve

200

mV

pH 7
Na+ /K+

50100

K+
pH 7

pH luminal 4.0 I outward
pH luminal 7.0 I outward

I outward

400

calculated before and after DCPIB application. B

200log [M]
0-5.0-200

Figure 2: A Bar graph of seal resistance

I inward

-4.5

-4.0

-3.5
pH luminal 4.0 I inward
pH luminal 7.0 I inward

Representative traces recorded in control and in
the presence of increasing concentrations of DCPIB,
using luminal solution with pH 4.0, and C pH 7.0. D
Concentration response curve of DCPIB application
using different luminal solution, with pH 4.0 (red) and7.0 (black); in both experiments, cytosolic solution
was pH 7.0.

Figure 3: A Representative TMEM175 traces
recorded in control and in the presence ofCytosolic solution

100 µM DCPIB (light green) and 2 mM 4-AP (darkgreen); pHluminal 7.0 – pHcytosolic 7.0. B Effect ofluminal solution exchange (from pH 7.0 toA

800

in control and in the presence of 100 µM DCPIB

4-AP
pH 7.0pH 7.0 4-AP 2 mM
pH 7.0 DCPIB 100 µM
pH 7.0 vehicle

I TMEM175 (pA)

600

pH 4.0) on TMEM175 current in the presence of
4-AP. C Representative TMEM175 traces recordedvehicle DCPIB

Luminal solution

pH 4.0B

800

400-100

400200mV-50

pH 4.0 4-AP 2 mM
pH 7.0 4-AP 2 mM

I TMEM175 (pA)

600

200

mV50-200100H-100+-50-400

50-200100H+-400(light green) and 2 mM 4-AP (dark green); pHluminal
4.0 – pHcytosolic 7.0. D Effect of luminal solution

Cytosolic solution

exchange (from pH 4.0 to pH 7.0) on TMEM175vehicle DCPIB

Luminal solution

4-AP
pH 4.0pH 7.0current in the presence of 4-AP.

C800

pH 4.0 4-AP 2 mM
pH 4.0 DCPIB 100 µM
pH 4.0 vehicle

I TMEM175 (pA)

600

D800

400-100

400200mV-50-200200mV50

100

H+-100-50-400Conclusions

pH 7.0 4-AP 2 mM
pH 4.0 4-AP 2 mM

I TMEM175 (pA)

600

-200

50100

H+-400For the first time we report recordings from freshly isolated
lysosomes on a high throughput automated patch clamp
device, the SyncroPatch 384.
Our method satisfies the needs of primary lysosomal recordings

The results clearly indicate that the combination of ourat high throughput to explore the pharmacology of lysosomal

Organellar Chips/ultra low cell density method should provide

channels in the native system. This allows a more detailed

efficient and low cost means of electrophysiological access tounderstanding of the function of lysosomal channels as well

not only lysosomal channels, but also to various ion channels inas their role as therapeutic target for numerous diseases

other types of organelles and intracellular compartments.

circumventing possible caveats that stem from the detour ofheterologous expression systems in the plasma membrane or

It further highlights the flexibility of the SyncroPatch 384 to beusage of indirect recording approaches. We have applied ouradaptable and adjustable to all needs for patch clamp andapproach to record currents from lysosomes overexpressing

allows lysosomal recording of high quality in a high throughput

TPC2 as well endogenous TMEM175 currents where we usedfashion similar to what has been observed from whole cells.

specific pharmacology and tight quality control to confirm thevalidity of our recordings.

B
Median Rseal (Ohm)A

Activation of TPC2 by 5µM TPC2_A1P Rseal
2,00E+09
1,60E+09
1,20E+09
8,00E+08
4,00E+08
0,00E+00

CD
1E-09

5E-10

Current (nA)

Current (nA)

1E-09

0E+00
-5E-10-1E-09Start of Recording (unstimulated)
End of Recording (stimulated with 5µM TPC2)
End of Recording (Vehicle only)+5 µM TPC2-A1-P

Figure 4: A Lysosomal preparation used for the5E-10

recordings from HEK cells stably overexpressing

0E+00-5E-10-1E-09

-2E-09

-2E-09

-2E-09

-2E-09+Reference solution

TPC2-YFP, scale bar indicates 10 µm. B Median
Rseal at start (n=69) and end of recording when
only reference solution was applied (n=15) whereas
reduction in Rseal during TPC2 activation (n=54).
C-D Exemplary current voltage ramps of Na+currents through TPC2 in presence or absence of5 µM TPC2_A1_P.

Figure 5: A Example lysosomal recording applying

A3 different concentrations of TPC2_A1_P as

B

2E-09

Current (nA)

Current (nA)

5E-10

-5E-10-2E-09
-2E-09

C-5E-04

0E+00-1E-09indicated. B Example current-time plot of0E-00

1E-09+0.5 µM TPC2-A1-P+5.3 µM TPC2-A1-P+27.6 µM TPC2-A1-P-2E-0330 s+5.3 µM TPC2-A1-P-4E-03-4E-03

inward current from recordings such as in A.
Concentration response data for TPC2_A1_P at-100 mV C as well as outward current at +80 mV-3E-03-3E-03D-3E-09+0.5 µM TPC2-A1-P-1E-03-2E+03

TPC2+27.6 µM TPC2-A1-P

D yielding an EC50 of 5.4 ± 1.6 µM (n=32) and 6.6 ±2.7 µM (n=32), respectively.

Methods

References
1. Xu, H., et al. Cell Calcium. 2015 Jul;58(1):1-10. doi: 10.1016/j.
ceca.2015.02.006.

Cells/Lysosomal Isolation
HEK-293 cells expressing TPC2-YFP was kindly provided fromProf. Grimm 7-9 and Lysosomes were isolated according to

2. Jinn, S., et al. Hum. Mol. Genet. 2019 Oct 1;28(19):3244-3254.
doi: 10.1093/hmg/ddz136. 3).
3. Hopfner, F., et al. Mov. Disord. 2020 Jul;35(7):1245-1248. doi:
10.1002/mds.28037.
4. Abeer, F., et al. Cell Chem. Biol. 2021 Vol 28, Issue 8.
5. Bazzone, A., et al. Int. J. Mol. Sci. 2023 Aug 14;24(16):12788.
doi: 10.3390/ijms241612788.

Schieder et al. 5. HEK-293 cells were used for the investigationof endogenous TMEM175; lysosomes were isolated using aprotocol developed and customized at Axxam.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch 384 employing the ultra-low cell density
approach and using Nanion’s Organellar Chips.

6. Hu, M., et al. Cell 2022 Jun 23;185(13):2292-2308.e20. doi:
10.1016/j.cell.2022.05.021.
7. Zheng, W., et al. Sci. Adv. 2022 25 Mar Vol 8, Issue 12 DOI:
10.1126/sciadv.abm1568.
8. Schieder, M., et al. Sci. Signal. 2010 Dec 7;3(151):pl3. doi:
10.1126/scisignal.3151pl3.
9. Schieder, M., et al. J. Biol. Chem. 2010 Jul 9;285(28):2121922. doi: 10.1074/jbc.C110.143123.

TMEM175: Traces were recorded from a holding potential of
0 mV, a voltage protocol from -100 to +100 mV with 1 s
duration, was applied every 4 s to evoke TMEM175 current. Hillequation was used for EC50 calculation.
TCP2: lysosomes were held at +60mV, a voltage ramp protocol
from -100 to 80 mV in 1s was applied every 10s. To estimate
EC50 currents were normalized to maximum response and data
fitted using the Hill equation.

10. Chen, C-C., et al. Nat. Protoc. 2017 12:1639-1658.
11. Sakurai, Y., et al. Science 2015 347:995-998.
12. Grimm, C., et al. Nature Commun. 2014 5:4699.
13. Gerndt, S., et al. Elife. 2020 Mar 16;9:e54712.
14. Gerndt, S., et al. FEBS J. 2020;287(24): 5284-5293.

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info@axxam.com I www.axxam.com
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