Ion Channels:

Application Note

K V7.2/7.3
Cells:
HEK293

Cross-site study of neuronal KV7.2/7.3
heteromers on the Patchliner

Tools:Patchliner

Nanion Technologies GmbH, Munich and ApconiX,
Alderley Park, UK

Summary
The KV7 channels are a family of voltage-gated potassium

the market in 2017 due to adverse side effects. In addition

the KCNQ1-5 genes1. The channels exist as tetramers, withtarget to treat pain, neuropsychiatric disorders such as anxiety,ion channels with five members (KV7.1 - 7.5) encoded byto epilepsy, KV7.2/7.3 has also been proposed as a potential

each subunit containing six transmembrane domains withattention deficit–hyperactivity (ADHD), mania, bipolar disorder

cytoplasmic N- and C-termini. The long intracellular terminus

and schizophrenia

is essential for tetramermization as well as interaction withcritical regulators such as PIP2, calmodulin, protein kinase Cand ankyrin G2. KV7-mediated currents are voltage activated,slowly activating and non-inactivating and are involved

in repolarization of the cell membrane potential, thereby
controlling cell excitability3. KV7.1 channels are primarily

expressed in cardiac cells whereas KV7.2, KV7.3 and KV7.5

are widely distributed in neuronal and primary sensory cells2.
KV7.2/7.3 heteromeric channels primarily underlie the neuronal
M-current (IKM)which plays a crucial role in repolarizing

neuronal membrane potential after a depolarizing input which
limits repetitive firing and is, therefore, a key mechanism inspike frequency adaptation4.
Mutations in KCNQ2 and KCNQ3 have been shown to cause
benign familial neonatal convulsions (BFNC), a rare inherited

Results
KV7.2/7.3 stably expressed in HEK293 cells were used on thePatchliner at two different sites, Nanion HQ and ApconiX.

Current responses to increasing voltage steps were used toconstruct current-voltage plots. The curves were fit with aBoltzmann equation and the IV plot for an average of 18 cells
recorded at ApconiX is shown in Figure 1A with traces from an
example cell shown in the inset. The average Vhalf calculated
from 18 individual plots was -19.9 ± 2.0 mV in excellent

agreement with the literature6. At a second site (Nanion HQ),
the average Vhalf calculated from 16 individual plots was -22.2 ±1.8 mV showing excellent consistency across the two sites. Therecordings were also highly stable with little change in peak
amplitude over a 15 minute measurement period. Figure 1B

form of epilepsy, and neonatal epileptic encephalopathy
dependence of the resulting channel, alter the interaction

A2.5

with PIP2, affect the subcellular localization at key neuronal

2.0

sites or cause loss of channel function2. Activation of KV7.2/7.3

Imax (nA)(NEE)2,4. Mutations in the gene can affect voltage

1.5is, therefore, a potential strategy to treat epilepsy with

1.0

retigabine (ezogabine) approved as an adjunct therapy inpartial-onset seizures in 2011, but was later withdrawn from

0.5-60-40

KV7.2/7.3
V0.5 Act = -19.9 ± 2.0 mV(n = 18)-20
020Voltage (mV)40

0.5 nA
0.1 s60

BFigure 1: A Current-voltage plot for KV7.2/7.3
with representative traces. B Stability of KV7.2/7.3
recordings. Currents could be recorded for over 15minutes with little change in amplitude over time.
Shown are raw traces from an exemplar cell andthe corresponding time course in external solution.

Imax (nA)

0 nA

2 nA

10864200200

Time (s) 400

600200 ms800

Figure 2: A Amitriptyline inhibited KV7.2/7.3 in a

A 1.0% inhibition

Amitriptyline
IC50 = 10.1 ± 1.3 µM (n = 22)
0.8 Hill = 1.7 ± 0.2

Controlconcentration-dependent manner. The CRC

1 µM
3 µM
10 µM

0.6

for an average of 22 cells is shown and tracesfrom an example cell. B Retigabine enhanced

30 µM
100 µM

0.4

KV7.2/7.3-mediated responses. The CRC for an
average of 5 cells is shown and example tracesfrom one cell +/- retigabine.

0.20.010 nM 100 nM

1 nA

1 µM 10 µM 100 µM

50 ms[Amitriptyline]

B 1.0Retigabine
EC50 = 19.7 ± 2.5 µM (n = 5)

0.8

Control
100 µM Retigabine

I/Imax0.60.40.2
0.0

0.5 nA
110[Retigabine] (µM)

100

50 ms

shows the timecourse and corresponding raw traces from anexample cell.
Amitriptyline is a tricyclic antidepressant with analgesic andsedative properties, although it can be associated with severeside effects such as seizures7. It blocks both KV1.1 and KV7.2/7.3

channels which could underlie the neuroexcitatory side effectsof the drug7. Amitriptyline blocked KV7.2/7.3-mediated currents
with an IC50 at one site (Nanion HQ; Figure 2A) of 10.1 ± 1.3 µM(n = 22) and at the second site (ApconiX) of 6.9 ± 1.2 µM (n = 8)
in excellent agreement with each other (P>0.05, unpairedStudent’s t test) and with the literature7.
Retigabine is an anticonvulsant that enhances KV7.2/7.3-

mediated responses by shifting the voltage-dependenceof activation to more hyperpolarized potentials8,9,10. In ourexperiments, retigabine enhanced KV7.2/7.3-mediatedresponses in a concentration-dependent manner with an
EC50 = 19.7 ± 2.5 µM (n = 5). Figure 2B shows the concentration

response curve (CRC) for an average of 5 cells and the traces
for an example cell in the presence and absence of 100 µM
retigabine.
In summary, KV7.2/7.3 expressed in HEK cells was recordedon the Patchliner at two different sites with very comparable
results for Vhalf and pharmacology. The Patchliner is anexcellent tool for studying KV7.2/7.3 and identifying novel

anticonvulsants and analgesic compounds which enhance
KV7.2/7.3 activity.

References

3. Tatulian, L., et al. 2001. 21(15):5535 – 5545
4. Rogawski, M.A. TINS. 23(9): 393 - 398
5. Wulff, H., et al. 2009. Nat. Rev. Drug Discov. 8: 982 - 1001
6. Gamper, N., Stockand, J.D., & Shapiro, M.S. 2003. 23(1): 84– 95
7. Punke, M.A., & Friederich, P. 2007. Anesth. Analg. 104(5):1256 - 1264
8. Main, M.J. et al. 2000. Mol. Pharmacol. 58: 253 – 262
9. Wickenden, A.D., et al. 2000. Mol. Pharmacol. 58: 591 – 600

Methods
Cells
HEK293 cells stably expressing KV7.2/7.3 were used.
Electrophysiology measurements
Cells were cultured and harvested according to Nanion’s
standard protocols. Cells were resuspended in external
recording solution and stored in the CellHotel of the Patchliner
before being dispensed into each well of the NPC-16 chip.
Internal and external solution compositions are available uponrequest. For IV plots, voltage was increased in 10 mV steps
from -60 mV to 60 mV from a holding potential of -80 mV. For
amitriptyline, a single step to 60 mV from a holding potential of-80 mV was used and for retigabine, a single step to 0 mV wasused. Data was analyzed in Igor or Prism. IV plots were fit with aBoltzmann equation and CRC plots with a Hill equation. Datais given as mean ± S.E.M.

1. Jepps, T.A., et al. 2021. Front. Physiol. 12:679317
2. Barrese, V., Scott, J.B., & Greenwood, I.A. 2018. Annu. Rev.
Pharmacol. Toxicol. 58: 625 - 48

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