Application Note

Channel:Cells:
Tools:

hTMEM16A
HEK293
SyncroPatch 384PE

Internal perfusion of Ca2+ to activate
TMEM16A/ANO1 on the SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by SB Drug Discovery.

SummaryResults

TMEM16A/Anoctamin1channel(CACC)

isa

which

has−CACCs such as TMEM16A are activated by cytosolic

functional

calcium and activation is influenced by membrane

Ca -activated
2+abroadCl

spectrum in processes including trans-epithelial ion

potential1,3,4.

transport, olfaction, photo-transduction, smooth muscle

internal solution must be exchanged from a Ca2+ freecontraction, nociception, cell proliferation and control ofsolution to a solution containing Ca2+. On the SyncroPatch

neuronal excitability . TMEM16A has been implicated to384PE the internal perfusion feature was used to activate

play a role in a number of health disorders and may be

hTMEM16A using internal application of Ca2+. The

an important therapeutic target in cystic fibrosis, asthma,current was then blocked by external application ofpain and some human cancers1.

niflumic acid (NFA; Figure 1).

1

TMEM16A

isactivatedbyelevatedTherefore,

toactivate

TMEM16A

thecytosolic

calcium concentrations . In conventional patch clamp
1

experiments, exchange of the intracellular solution toinclude calcium in order to initiate channel activity ischallenging and typically performed using inside-out
patches or by comparing the effect of internal calcium
between different cells in the whole cell configuration.
Furthermore, current run-down or desensitization arecommon problems associated with recording this ionchannel2.
Here we present data from HEK293 cells expressinghTMEM16A in whole cell and perforated patch modeusing fluoride-free internal solution on the SyncroPatch 384PE. The data show that intracellular solution canbe exchanged in a very robust manner to investigate
calcium

sensitivity,

voltagedependence

andpharmacology of the channel.

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Figure 1: 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 over 1 s (intersweep holding potential -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) areplotted versus time. The application of internal Ca2+ is indicated by thered bar. Three minutes of baseline current was recorded prior to internalexchange. During the experiment extracellular buffer was applied twice(white region), followed by reference buffer (light blue region) and at theend a full-block concentration of 250 µM NFA was added (darker blueregion). The current increased upon application of internal Ca2+ reachinga peak within 1- 2 min after the start of perfusion. The current was stable
and completely inhibited by NFA. The recording was performed in thewhole cell configuration using a single hole chip.

Application Note

10I (nA)

0.4 µM
1.7 µM
4 µM
15.7 µM

864
2-100

-50

-250
Voltage (mV)

100

Figure 3: hTMEM16A is Ca2+ and voltage-dependent. Four different
concentrations of Ca2+ were used to activate hTMEM16A in parallel on asingle plate using the perfusion system of the SyncroPatch 384PE. A fourstep voltage protocol (-100mV, -20mV, 40mV and 100mV) was applied
every 15 s. The current-voltage plot shows that current amplitude increases
in response to increasing Ca2+ concentration and by increasing ΔV. The
recording was done in perforated patch mode on a single hole chip.

Figure 3 shows that hTMEM16A activation is both Ca2+and voltage-dependent as expected1,3,4. In this experiFigure 2: Representative Online Analysis using different concentrations ofCa2+ for activation. hTMEM16A was activated by internal perfusion andextracellular solution was applied twice (grey lines; RefSol). The current
amplitude at 100 mV (dark blue) and -100 mV (light blue) is plottedversus time. Full block with NFA was performed at the end. Using 1.7 µM
intracellular Ca2+ the channel was activated which was followed by an
immediate decrease in current. The remaining smaller current was stable
over time. 4 µM intracellular Ca2+ activated TMEM16A currents without
any current decrease resulting in a robust and stable current response. At
the highest concentration (15.7 µM ) rundown was evident and the assaywindow was less stable.

In order to investigate the optimal concentration ofinternal Ca

2+ment, the internal solution was exchanged from 0 Ca2+ toCa2+-containing solution whereby six columns of the chipreceived one concentration (0.4, 1.7, 4 or 15.7 µM) of Ca2+and voltage was varied using a 4-step voltage protocol.
Figure 4 shows the concentration response curvesof NFA at different membrane potentials. NFA is lessefficaciousatnegativepotentials.

Thisfinding

isconsistent with published results5,6. The IC50 values are ingood agreement with those reported previously5,6.

to reliably activate hTMEM16A, different

concentrations of Ca2+ were used. Figure 2 shows thecurrent amplitudes at 100 mV (dark blue) and -100 mV(light blue)versus time for cells perfused with 1.7 µM,
4 µM or 15.7 µM internal free-Ca2+. Using 1.7 µM Ca2+,hTMEM16A-mediatedcurrents

wereactivatedbutquickly declined by approximately 30% after which thecurrent remained stable during two applications ofreference solution (external). A higher concentrationof Ca2+ (4 µM) produced best results, with robust andstable

activationofhTMEM16A

currents

whereasthe highest concentration tested (15.7 µM) resultedinlarge

pronounced

activationofrundown(>30%)

thecurrent

overthebut
courseof the experiment. For this reason, we used 4 µM
Ca2+toactivate

hTMEM16A

for

pharmacologicalexperiments.

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Figure 4: Concentration response curves of NFA on hTMEM16A at different
voltages. The concentration response curves were calculated across theplate using a full block concentration (250 µM NFA) at the end of theexperiment for normalization. IC50 at -100 mV = 17.9 µM, IC50 at -20 mV =7.6 µM, IC50 at 40 mV = 6.1 µM (not shown), IC50 at 100 mV = 4.7 µM. The
recording was performed in perforated patch mode on a single hole chip.

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depiction of online analysis
data of hTMEM16A expressing HEK293 cells as recorded on one NPC-384 (single-hole) patch clamp chip. Three hundred and eighty-four small color-coded
pictures as seen in the upper left part display 384 recordings. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayedon the right. Graphs show current amplitudes following internal Ca2+ (4 µM) activation (white region), followed by block by different concentrations of NFA(blue region; concentrations indicated) and finally full block with 250 µM NFA (grey region).

Exp end and chip
unload5 min

10 - 15 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash, activation using
internal perfusion of Ca2+, 2 compound additions(compound of interest, full block)

Analysis

Figure 6: The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a single point concentration response curve plus
activation of hTMEM16A with internal Ca2+ and full block at the end of the experiment took approximately 17-22 min.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384

Different internal solutions were used to activatesoftware during an experiment. The user can choose

hTMEM16A on a single plate to investigate Ca2+

whether to visualize raw traces or online analysis. Independence. The timeline of each experiment was

this case,online analysis is chosen and the graphs

about 17-22 minutes (start – end; Figure 6) and included

represent current amplitude plotted against time. An

wash, activation with internal Ca2+, time to reach a stable

individual well can be highlighted to monitor thecurrent, single compound concentration application

progression of the experiment. In the Online Analysis

and full block, all performed on each individual well,view, the time points at which solution additions havedrastically reducing the consumable cost per data point

been made are indicated by vertical lines, as well as

to < $0.60.

different background colors. In this case, white shows
activation of hTMEM16A by internal Ca2+, blue shows

The SyncroPatch 384PE is a high throughput and highly

inhibition by different concentrations of NFA and grey

reliableshows full block with NFA.

recording hTMEM16A currents. User-friendly software,automatedpatchclamp

deviceforexcellent success rates, single additions or multiple
In conclusion, hTMEM16A expressed in HEK293 cells canbe recorded on the SyncroPatch 384PE using fluoridefree internal solution. The internal perfusion feature of
the SyncroPatch 384PE was used to activate hTMEM16A

additions of compound to each cell, perfusion of theinternal solution and easy analysis result in reliable highquality data at an increased throughput with an
economical cost per data point.

using internal Ca2+ and increase in the current could bemonitored
current

online.

amplitude

Thisiscontinuous
importantformonitoringof

distinguishingcompound effect from increase in leak or loss ofseal and can be a benefit for assay development.

Electrophysiology

References
1. Pedemonte, N. & Galietta, L.J.V., 2014. Physiol. Rev. 94: 419-45.
2. Ni, Y-L., et al. 2014. PLoS ONE 9(1): e86734. doi:10.1371/ journal.
pone.0086734.

Whole cell and perforated patch clamp recordings wereperformed according to Nanion’s standard procedure
for the SyncroPatch 384PE using single hole chips and F-free internal solution. The concentration of free Ca2+ was

3. Arreola, J., et al. 1996. J. Gen. Physiol. 108: 35-47.
4. Kuruma, A. & Hartzel, H.C. 2000. J. Gen. Physiol. 115: 59-80.
5. Bradley, E., et al. 2014. Channels. 8(4): 308 - 320.

estimated using the CaBuf prediction software from Guy
Droogmans, Department of Physiology, KU Leuven,Belgium. Currents were recorded using a voltage ramp

6. Liu, Y., et al. 2015. Pflugers Arch. 467(7): 1417-1430.

protocol from -120 mV to +60 mV over 1 s (intersweepMethods

holding potential -60 mV) or voltage step protocolCells

each for 200 ms and with 2 s at the holding potential ofinvolving 4 steps to -100 mV, -20 mV, 40 mV and 100 mV

hTMEM16A expressing HEK293 cells were supplied by SB-60 mv between each step. This was repeated every 15 s.

Drug Discovery, Catalogue ref: SB-HEK-Ano1.
Cell culture
Cells were cultured according to the manufacturer’s
recommendations and harvested according to Nanion’s
standard protocol using TrpLE.

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fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960