Application Note

Channel:Cells:
Tools:hTRPC5
HEK293
SyncroPatch 384i

Internal perfusion of Ca2+ to activate hTRPC5
on Nanion’s SyncroPatch 384PE
The

electrophysiology

teamatNanion

Technologies

GmbH,Munich.

Cells kindly provided by Charles River.

SummaryResults

Transient Receptor Potential Canonical (TRPC) channels

For the evaluation of the performance of hTRPC5 (HEK293)

are a subfamily of TRP channels. The TRPC family contains

cells, Seal Resistance (Rseries) at the start and end of one

at least 7 subunits and are predominantly expressed inexperiment using single hole chips was determined (Fig.

neuronal cells where they may play an important role in1). The seal resistance improved over the course of theCa flux .

experiment with 64% of cells with a seal resistance >0.52+1GΩ at the start of the experiment increasing to 77% at
TRPC5 channels are non-selective cation channels

the end of the experiment which lasted approximately

expressed in many areas of the brain particularly the10 minutes.

hippocampus, amygdala and cerebellum, amongst
patho-70

physiological role of TRPC5 is not fully known, it does60andappear to be important in neuronal function, in particcampal neurite outgrowth and growth cone morphol. Knockout mouse studies have also revealed thatTRPC5 plays an essential role in innate fear5. TRPC5 is40
30

expressed in some regions outside of the CNS including20

the heart where it contributes to cardiac hypertrophy in

10

2heart failure6.

by 2-APB with an IC50 consistent with that reported in theliterature10.

Download more Application Notes from www.nanion.deG1>

G-1hm

OOhmhmsabldi50
0lanthanide, gadolinium (Gd3+), as expected9 and blocked

catc
h/

contain free-Ca2+. This current was potentiated by theobe activated by perfusion of the intracellular solution toN

SyncroPatch 384PE, TRPC5 expressed in HEK cells couldO

TRPC5 is activated by intracellular calcium . Using thehm

ed

07,8O

ogy

1,3,4

50% Cells

ular during development where it is involved in hippo-

start of experimentend of experimentM

physiologicalM

the00Although<5others2.

Seal Resistance
Figure 1: Statistics of TRPC5 expressing HEK293 cells recorded onone NPC-384 chip on the SyncroPatch 384PE. Success rate (sealresistance) of individual HEK cells on the SyncroPatch 384PE.
Shown is a bar graph of seal resistances at the start (light blue)and end of the experiment (dark blue).

Application Note
Currents mediated by hTRPC5 were activated byapplication of 150 nM free-Ca2+ in the internal solution.
The timecourse of activation of the current followinginternal perfusion of Ca2+ could be monitored online andis shown in Figure 2. There is a gradual increase in current(at 90 mV) over time, peaking approximately 2 min after
the start of internal Ca2+ perfusion. The TRPC5 current wasblocked by application 50 μM 2-APB (full-block concentration). All experiments utilized 150 nM internal free-Ca2+and were performed using multi-hole (4 holes per well)chips in the whole cell configuration.

Figure 3: Average concentration response curve for 2-APB. The concentration
response curve was constructed across the whole plate. Only cells which
returned to baseline after the full block concentration of 2-APB were usedfor analysis giving an IC50 = 10.8 μM (n = 330) and a success rate of 86% forcompleted experiments.

TRPC5 is modulated by the lanthanides9, La3+ and Gd3+.
Gd3+(100 μM) potentiated the TRPC5 current activated byinternal Ca2+, particularly the inward current at -90 mV as
previously reported9. Figure 2: Activation of TRPC5 by internal perfusion of 150 nM free-Ca2+. Online
analysis (current amplitude at 90 mV) is plotted versus time and applicationof internal Ca2+ is indicated by the red bar. At the end of the experiment,a full-block concentration of 2-APB (50 μM) was applied (grey region).
The current increased upon application of internal Ca2+ reaching a peak
approximately 2 min after the start of perfusion. The current was stable andthen blocked by external 2-APB.

TRPC5 activated by elevated internal Ca2+ was blocked
by external 2-APB in a concentration-dependent manner.
In these experiments, the cells were activated by internal
perfusion of 150 nM internal free-Ca2+ following which
the cell was exposed to a single concentration of 2-APB
applied externally and the concentration response curve
calculated across the whole plate. A second (full block)concentration of 2-APB (50 μM) was also applied acrossthe whole plate as a positive control. The average
concentration response curve for 330 cells is shown in
Figure 3. The calculated IC50 was 11 μM (n = 330) which
is similar to that reported in the literature (19 μM)10. In thisexperiment, 330 cells were used for the concentration
response curve giving a success rate of 86% forcompleted experiments.

Download more Application Notes from www.nanion.de

Figure 4: TRPC5 is potentiated by Gd3+. A Raw traces from an exemplar cellin the presence of internal Ca2+ and potentiation of the response following
application of external Gd3+ (see black trace). B Corresponding online
analysis showing current amplitude at 90 mV (dark blue squares) and -90 mV(light blue circles) in the presence of internal Ca2+ (white), 100 μM external
Gd3+ (blue) and full block by 50 μM 2-APB (grey).

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depictionof online analysis data of hTRPC5 expressing HEK293 cells as recorded on one NPC-384 (multi-hole, 4 holes per well) 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 displayed on the right. Graphs show current amplitudes of internal
Ca2+ (150 nM) activation (white region), block by 2-APB (blue region) at the indicated concentrations and full block (grey region) using
50 µM 2-APB. Five minutes of baseline current was recorded prior to application of internal Ca2+. Only cells which returned to baseline
after the full block concentration of 2-APB were used for analysis.

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 TRPC5 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 NoteFigure 5 shows a screenshot of the SyncroPatch 384

was about 17-22 minutes (start – end) and included wash,

software during an experiment. The user can choose

activation with internal Ca2+, time to reach a stable

whether to visualize raw traces or online analysis. In

current, single compound concentration application andthis case,online analysis is chosen and the graphs

full block, all performed on each individual well, drasticallyrepresent current amplitude plotted against time. An

reducing the consumable cost per data point toindividual well can be highlighted to monitor the< $0.60.

progression of the experiment. In the Online Analysis view,the time points at which solution additions have been

The activation properties of hTRPC5 recorded on themade are indicated by vertical lines, as well as different

SyncroPatch 384PE are in excellent agreement withbackground colors. In this case, white shows activation

those reported in the literature8,9,10. As expected, hTRPC5of TRPC5 by internal Ca2+, blue shows inhibition by thecould be activated by internal application of calcium7,8,indicated concentration of 2-APB and grey shows fullpotentiated by gadolinium9 and inhibited by 2-APB

block by 50 µM 2-APB.

in a concentration-dependent manner with an IC50
similar to that reported in the literature10.

In conclusion, hTRPC5 expressed in HEK293 cells can berecorded on the SyncroPatch 384PE with an excellent
success rate for completed experiments. The internalperfusion feature of the SyncroPatch 384PE was used
to activate TRPC5 and increase in the current could bemonitored online. This continuous monitoring of current
amplitude is important for distinguishing compound effectfrom increase in leak or loss of seal and can be a benefit
for assay development. The timeline of each experiment

The SyncroPatch 384PE is a high throughput and highly
reliable

automatedrecordinghTRPC5patchcurrents.

clamp

deviceUser-friendlyforsoftware,excellent success rates, single additions or multipleadditions 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.

Cell culture

References

Cells were cultured according to the manufacturer’s

1. Sun, Y., et al. 2014. Cells. 3: 455-475
2. Riccio, A., et al. 2002. Mol. Brain Res. 109 95-104
3. Greka, A., et al. 2003. Nat. Neurosci. 6(8): 837-845
4. Abramowitz, J. & Birnbaumer, L. 2009. FASEB J. 23: 297328

recommendations and harvested according to Nanion’s
standard protocol using detachin.
Electrophysiology
Whole cell patch clamp recordings were conducted

5. Riccio, A., et al. 2009. Cell. 137: 761-772
6. Bush, E.W., et al. 2006. JBC. 281(44): 33487-33496
7. Okada, T., et al. 1998. JBC. 273(17): 10279-10287
8. Blair, N.T., et al. 2009. J.Gen. Physiol. 133 (5): 525–546
9. Jung, S., et al. 2003. JBC. 281(44): 33487-334969.
10. Xu, S-Z., et al. 2005. Br. J. Pharmacol. 145: 405-415

Methods

according to Nanion’s standard procedure for theSyncroPatch 384PE using either single- or multi-hole (4holes per well) chips. A voltage ramp protocol from -100
mV to +100 mV over 100 ms was applied to the cells every
10 s. Intersweep holding potential was -20 mV. Current
amplitude at +90 mV and -90 mV was used for analysis.
Compounds were applied via the internal and externalsolutions.

Cells
hTRPC5 expressing HEK293 cells were supplied by CharlesRiver.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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