lnvestigation of the Ion Channels hTMEM16A/Ano1 andTRPC5 and their Modulation by Intracellular Calcium
Nina Brinkwirth1, Tom Götze1, Markus Rapedius1, Søren Friis1, Andrea Brueggemann1, Tim Strassmaier1, Claudia

Haarmann1, Ilka Rinke1, Marius Vogel1, Timo Stengel1, Johannes Stiehler1, Michael George1 and Niels Fertig1
1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany, contact: Info@nanion.deAbstracthTMEM16A/Ano1
Internal Perfusion of Ca2+ to Activate the Channel / Inhibition by NFA

TargetshTMEM16A/Anoctamin1 is a Ca2+ activated Cl- channel (CACC) which has a broad functional spectrum inprocesses including trans-epithelial ion transport, olfaction, photo-transduction, smooth muscle contraction,
nociception, cell proliferation and control of neuronal excitability1. The ion channel has been implicated toplay a role in a number of health disorders and may be an important therapeutic target in cystic fibrosis,
asthma, pain and some human cancers1.
hTRPC5 is a non-selective cation channel expressed in many areas of the brain particularly the hippocampus,
amygdala and cerebellum, amongst others 2. Although the physiological and patho-physiological role ofhTRPC5 is not fully known, it does appear to be important in neuronal function, in particular during
development where it is involved in hippocampal neurite outgrowth and growth cone morphology 3,4.
Knockout mouse studies have also revealed that TRPC5 plays an essential role in innate fear 5. hTRPC5 isexpressed in some regions outside of the CNS including the heart2 where it contributes to cardiac hypertrophy
in heart failure6.

Precise Ca2+ concentration is important

Stable current over 10 min
hTMEM16A gating is influenced by internal Ca2+ andmembrane potential. To activate hTMEM16A theinternal perfusion system was used while applying
voltage ramps. Upon application of internal Ca2+
the current increased reaching a peak within 1-2
min after the start of perfusion. The current was
stable and completely inhibited by NFA.
AChallengehTMEM16A and hTRPC5 are activated by elevated cytosolic Ca2+ concentrations. To initiate channel activity
intracellular solution must be exchanged from a Ca2+ free solution to a solution containing Ca2+. Some of thecommon instruments for automated patch clamp are limited to do an internal exchange at all. But also for theother devices it is challenging because they rely on fluoride in the internal solution to perform proper seals.
Unfortunately fluoride has a strong tendency to precipitate with calcium, which makes it impossible to correctly
adjust a precise Ca2+ concentration needed for channel activation.
Furthermore current run-down or desensitization are common problems associated with recording hTMEM16A7
and hTRPC510.

B

4µM free Ca2+

Accomplishment
On the SyncroPatch® 384PE we developed two very reliable assays for hTMEM16A and hTRPC5 in the presenceof fluoride-free intracellular solutions using the internal perfusion feature. The presented data show that theintracellular solution can be exchanged in a very robust manner to investigate Ca2+ sensitivity.
Besides voltage dependence and pharmacology characteristics of hTMEM16A and hTRPC5 are presented on
this poster. The hTMEM16A assay was established using the PE/Felix wheatear the hTRPC5 assay was run on thePE/Biomek.

Activation of hTMEM16A by internal perfusion of 4 μM free Ca2+.
A Raw traces of an example cell to a voltage ramp from -120 mV to+60 mV in the absence (black trace) and presence (red trace) of 4
μM internal free Ca2+. B Current amplitudes at +60 mV (dark blue)and -120 mV (light blue) are plotted versus time. The application ofinternal Ca2+ is indicated by the red bar. During the experimentextracellular buffer was applied twice (white region), followed byreference buffer (light blue region) and at the end a full-block
concentration of 250 μM NFA was added (darker blue region).

PatchEngine
HTS for Automated Patch Clamp

IC50 of NFA depends on voltage
A 10-point dose response curve of Niflumic acid (NFA) wasrecorded on hTMEM16A using the SyncroPatch® 384PE
reaching a success rate of 87% (334 wells).

CIn order to investigate the optimal
concentration of internal Ca2+ toreliably activate hhTMEM16A,different concentrations of Ca2+
were tested on the same Chip.
1.7μM
Ca2+,
hTMEM16Amediatedcurrents
wereactivated but quickly declined byapproximately 30% after which
the current remained stableduringtwoapplicationsofreference solution (external). Ahigher concentration of 4µM Ca2+
produced best results, with robust
andstableactivationofhTMEM16A currents whereas thehighest
concentration
tested(15.7 μM) resulted in large
activation of the current but
pronounced rundown (>30%)
overthecourseoftheexperiment. For this reason, weused 4 μM Ca2+ to activate
hTMEM16A for pharmacologicalexperiments.

Representative Online Analysis using different concentrations of free Ca2+ for activation.
C hTMEM16A was activated by internal perfusion and extracellular solution was applied twice (greylines; RefSol). The current amplitude at 100 mV (dark blue) and -100 mV (light blue) is plotted versus
time. Full block with NFA was performed at the end

E

IC50 at 100mV = 5.2 µM
IC50 at 40mV = 7.9 µM
IC50 at -100mV = 14.7 µM

DNFA385 color coded depictions of data traces judgment of success rate
0500

250

125

62.5

31.3

15.6

7.8

3.9

1.9

0.98

0

7.8 µM

62.5 µM

7.8 µM

Highlighted time plot of 1 cel1
showing activation of hTMEM16Aand inhibition of NFA

Highlighted time plots of 16 cells
showing activation of hTMEM16A
and inhibition of NFA

Concentration response curves of NFA on hTMEM16A at different voltages.
D Graphical user interface of the screening and data analysis software used on the SyncroPatch®
384PE. Graphs show current amplitudes following internal Ca2+ (4 μM) activation (white region),
followed by block by different concentrations of NFA (blue region; concentrations indicated) andfinally full block with 250 μM NFA (grey region). E Plot of IC50 curves depending on voltage. IC50at -100 mV = 14.7 μM, IC50 at -20 mV = 9.6 μM (not shown), IC50 at 40 mV = 7.9 μM, IC50 at 100
mV = 5.2 μM.

Currents
wererecordedusingavoltage
step
protocol involving 4 steps to 100 mV, -20 mV, 40 mV and
100 mV each for 200 ms andwith 2 s at the holding
potential of -60 mv between
eachstep.
Thiswasrepeated every 15 s. The
protocol allowed to analyze
the IC50 of NFA at different
voltages.
The concentration response
curveswerecalculated
across the plate using a fullblock concentration (250 μMNFA) at the end of theexperiment for normalization.
The IC50 values are in goodagreement
withthose
reported previously8,9. NFA isless efficacious at negative
potentials. This finding is
consistent with publishedresults8,9.

SummaryhTRPC5
Potentiation by Gd3+ / Inhibition by 2-APB

 hTMEM16A (HEK293) and hTRPC5 (HEK23) were measured both successfully using the SyncroPatch®
 Compatible with different
liquid handler: Felix (CyBio),
Biomek FX (Beckman Coulter) 384 amplifiers, 384 pipetting head High gigaseal rate on HEK, CHO, LTK, RBL, iPS and RBC
 Fast solution exchange for ligand gated channels
 Partial usage of the chips possible (32 wells per run)
 Different cell lines can be used in one run (6, 12, 24)
 Temperature controlled cell hotel (4 - 70 OC)
 Customized chips for different hole sizes, allowing optimal

IC50 of 2-APB

hTRPC5 is activated by cytosolic Ca2+ and activation isinfluenced by membrane potential. Furthermore hTRPC5
can be potentiated by gadolinium (Gd3+). To study themodulation by Gd3+ hTRPC5 cells were activated byinternal Ca2+ while applying a voltage ramp. After awhile Gd3+ was added externally. A potentiation of thecurrent amplitude occurred immediately, particularly forthe inward current (-100 mV). This behavior stands ingood agreement with published results10. Finally thecurrent was fully blocked by 2-APB.

hTRPC5 was activated by elevated internal
Ca2+ and blocked by external 2-APB in aconcentration-dependent manner. A ramp
protocol was applied and the IC50 at 100mV
was calculated across the plate using a fullblock concentration of 50 μM 2-APB at the endof the experiment for normalization. Only cells
which returned to baseline after full block wereconsidered for analysis giving an IC50 of 7.59
μM and a success rate of 86% (328 wells). The
IC50 stands in nice agreement with reported
values in the literature11.

Fand data analysis

 Internal solution exchange

100mV

H

access and seal resistances for different cell lines

 Open design allows integration into robotic environments
 Upgradable current clamp option
 Upgradable temperature control option
 Complete, intuitive and flexible software for data acquisition384PE with two different liquid handlers; the Felix (TMEM16A) and the Biomek FX (hTRPC5).

TRPC5 is modulated by Gd3+

100ms-20mV

G-20mV-100mV

150nM free Ca2+

I+Gd3+

columns
be used in parallel• Multiple exchanges possible

channels activated by cytosolic Ca2+.

 The SyncroPatch® 384PE allows to monitore online the current increase during internal exchange, which
can be important for distinguishing compound effect from increase in leak or loss of seal and is a strong
benefit for assay development.

 Multiple internal solutions can be compared side by side. This way the Ca2+ concentration, which
delivers the most stable assay window for hTMEM16a, could be determined easily within one experiment.

 Both assays, for hTMEM16a and hTRPC5, provide a high and robust assay window, which allows to test
Inhibitors in a concentration depending manner. IC50 values were calculated across the plate using afull block at the end for a precise in-well-normalization. The IC50 values of NFA and 2-APB are in good
agreement with literature values.

 Excellent success rates of 87% (hTMEM16A) and 86% (hTRC5) were reached for IC50 experiments on bothtargets

 In conclusion our assay development demonstrates the ability of Nanion’s SyncroPatch 384PE to record

References:changed via 12 double

hTRPC5 is potentiated by Gd3+.
F Exemplary raw trace to a voltage ramp from -100 mV to +100 mV
activated with 150 nM Ca2+ in the absence (red trace) and presence(blue trace) of 100 μM Gd3+. Full block was applied at the end (50µM2-APB, black trace). G Corresponding Online Analysis showing current
amplitude at 100 mV (dark blue) and -100 mV (light blue) in presenceof internal Ca2+ alone (white region) and plus 100 μM external Gd3+(blue region). Full block with 50µM 2-APB (grey region).

 The internal perfusion system of the SyncroPatch 384PE is an indispensable feature for the investigation ofchallenging targets as hTMEM16A and hTRPC5 at a high throughput with excellent success rates.

• Internal solution is filled and• Up to 4 different solutions can The assays were stable and highly reproducible despite using fluoride-free internal solution.

Concentration response curve of 2-APB on hTRPC5.
H Voltage ramp protocol. I Plot of IC50 curve1.) Pedemonte, N. & Galietta, L.J.V., 2014. Physiol. Rev. 94: 419-45.
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