Investigating pain pathways by inhibition of voltage-gated sodium channels
E. Dragicevic1, N. Becker1, M. Rapedius1, T. Goetze1, I. Rinke-Weiß1, A. Obergrussberger1, S. Stölzle-Feix1, C. Haarmann1, R. Haedo2, M. George1, A. Brüggemann1, N. Fertig1
1 Nanion Technologies, Munich, Germany; 2 Nanion Technologies, Livingston, NJ

Abstract

State-Dependence of Tetracaine on hNav1.7 Channels

Voltage-gated sodium channels (NaV) are attractive targets for investigation ofchronic and neuropathic pain, due to their physiological role in action potential
generation and propagation and, thus, neuronal excitability. The NaV1.7 is foundprimarily in the peripheral nervous system and is thought to play a role in nociception
and pain sensing. The TTX-resistant NaV1.8 is selectively expressed in dorsal root
ganglion (DRG) neurons. We present recordings of NaV1.7 and NaV1.8 channels on ahigh throughput screening patch clamp platform.

Effect of Protoxin II on hNav1.7 channels

0.15 1.58 15.8µM Tetracaine

Current–voltage(IV)plotforhNaV1.7
recordedontheSyncroPatch 384PE for an averageof 275 CHO cells. Raw current tracesfrom a single cell are also shown.
Thrixopelma pruriens
Peruvian green velvet tarantula

1.

NaV1.7 was expressed in CHO cells and the current voltage relationship recorded
was consistent with NaV1.7 obtained using other methods. Vhalf of activation was -24
mV (n=275). Using a double-step voltage protocol we were able to investigate
whether compounds, such as tetracaine, exhibit state dependence. We show thattetracaine exhibited a lower IC on the second pulse, i.e. the inactivated state of thereceptor, compared with the resting state. NaV1.8 expressed in CHO cells started toactivate at approximately -40 mV, peaking at between 10 mV and 20 mV with aVhalf of activation of -2.7 mV (n=380). In order to study NaV channels involved in pain
pathways in a more physiological environment, we used stem cell-derived neurons,
more specifically with an overexpression of NaV1.8. In these cells, endogenous NaVmediated currents were recorded with activation parameters consistent with NaV1.7.

3.

2.

Amino acid sequences and structuresof spider-derived Nav1.7 blocker
protoxin-II (Pennington et al. 2017)4.

Protoxin-II inhibits both tetrodotoxin-sensitive and -resistant voltage-gated sodium channels, efficiently
blocking the human Nav1.7 channel. Here, selective inhibition of Nav1.7 channel IC50 by Protoxin-II wasmeasured in SyncroPatch 384PE. Currents were evoked by depolarizing pulses from a holding potential of -100
mV to step potential varying from -60 to 60 mV. The IC50 value for current inhibition by Protoxin-II was 1.56 nM.

hNav1.8 Current-Voltage (IV) Relationship
Recorded with Multihole Substrate

Our results demonstrate that NaV channels can be successfully studied on highthroughput electrophysiological systems, facilitating the discovery of novel pain
therapeutics.

Double voltage step protocol: -120 mV to 0 mV.
0 mV

Nav1.8
Nav1.7 ThresholdNav1.9Resting
Potential

1 ms

Modified from Dib-Hajj, et al, 2002. Trends in Neurosciences. 25(5): 253-259

Screenshot of the PatchControl 384PE software showing an experiment using CHO
cells expressing hNaV1.7. Peak current amplitude of the first peak (light blue) and thesecond peak (dark blue) is plotted against time. First, control solution without
tetracaine was added (white), and then increasing concentrations of tetracaine indifferent shades of blue (cumulative concentration response). Bar graph showing
seal resistance at the start (dark blue) and end (light blue) of the experiment. Here,
95% of cells had a seal resistance of >500 MΩ at the beginning, and 91% at the endof the experiment (after 20 min).

Analysis of tetracaine block of hNaV1.7 expressed in CHO cells using DataControl
384. Results file is loaded into DataControl 384 and sorted by compound name (1).
Average concentration–response curve (CRC) for tetracaine block of the firstpeak (2) and the second peak is shown (3). DataControl 384 software shows theraw data traces, online analysis, and concentration–response curves for eachindividual cell. The concentration–response curves for the first and second peaks
are displayed overlaid (4).

Vhalf (mV)

4x Chip
9x Chips
ManualData from Obergrussberger, et al, 2016. JALA. 21(6) 779–793-8.6(n=382)-12.8(n=381)-15.7Vhold (mV)

Cell type-100

CHO-100-100

CHO
DRG

These values agree well with theliterature (Cummins and Waxmann.

Stem Cell-Derived Neurons
Recorded on the Patchliner

1997. J.Neurosci., 17(10):3503-14).

High Throughput Automated Patch Clamp of hNaV1.9

Bar graphs (left): comparison of current levels obtained from single- and multihole (4x, 9x) chips
from similar recordings. Screenshot depicts NaV1.8 IV currents recorded with 9x multihole chip.

90% success rate

hNav1.9 is TTX-Insensitive

Compound Affinity and Assay Stability for theSlow Inactivated State of hNav1.8 Channels

QCs: R >100MΩ, I > 0.2nA (4x)

Step protocol

30ms
10mV
C1-30mV

Adherent iCell Neurons (left)
and in suspension for use on thePatchliner (right).

-100mV

1µM TTX
RefSol

RefSol

NaV-mediated currents recorded fromiCell Neurons were blocked by TTX withan
IC50
=12±6nM(n = 5) revealing a TTX-sensitive current
in these cells.

5 µM

25 µM

50 µM

C2The figure (left) shows a screenshot of the dataacquisition displaying OA results and theoverlaying current amplitudes in the absence
and presence of 1 µM TTX, followed by a blockof 1mM Lidocaine (full block)

1mM
Lidocaine

Incubation at 32°C Increases Channel
Expression

Concentration-Dependent Block of hNaV1.9 by
Lidocaine
25µM

50µM

2,0I median [nA]

250µM

500µM

2.5mM

A five point CRC for
Lidocaine block ofhNaV1.9
wasconstructed across theplate.

Current amplitudes of hNav1.9 with previous cellincubation at 32°C for varying lengths of time

1,6C1125 µM

250 µMTime course of current levels from recording on the left, applying
the two state pulse protocol. A 10-point CRC curve of tetracaine
was measured 6 times (dark blue rectangles) together with single
points for IC50 (light blue, 25 µM) and full block (blue, 250 µM)
from a 384 well compound plate.
Screenshot of a representative recording of hNav1.8 channels using
the voltage protocol shown on the left to asses the resting (C1) andslow inactivated state (C2). Currents are leak subtracted, holding
potential was -100 mV and pulse was applied once every 60s.
Average CRC for tetracaine (n=80/82) fitted with a standard Hillequation highlighting a ~16 fold shift for the IC50s obtained from theslow inactivated compared to the resting state. Bottom right graphshows a distribution of data points obtained from DMSO controls wells(n=291/302) which shows little rundown and highly consistent results.

C2 (slowinactivated)
IC50:2.22 µM
Hill:0.51
n=80

C1 (resting)
IC50:35.6 µM
Hill:1.01
n=80

1,2Summary

0,8✓ hNav1.7, 1.8 and 1.9 were recorded on the SyncroPatch 384PE with high success rates for Rseal, current levels and0,4Action potential elicited using an
1 ms current pulse of 80 pA. Action
potential was abolished by 10 µM
lidocaine
andreturned
uponwashout.

1 µM

100ms

As expected, hNav1.9 currents are TTX-resistant.

Data from Haythornthwaite, et al, 2012.
JBS. 17(9) 1264-1272
Current responses to a voltage step
protocol of Dopa.4U Neurons (left)
and the corresponding IV plot for an
average of 5 cells (right). Vhalf was -31
mV when calculated using aBoltzmann equation.

1µM TTX

0.5 µM//

100ms

DMSO-Controls

0.25 µM30ms
C2Sodium channels are classified according totheir sensitivity to the neurotoxin TTX. They areseparated into two classes, TTX-sensitivechannels (e.g. NaV1.7) and TTX-resistantchannels (e.g. NaV1.8 and NaV1.9).

1mM
Lidocaine

Current responses to a voltage step protocol of iCell
Neurons (left) and the corresponding IV plot for an
average of 54 cells (right). Vhalf was -20 mV whencalculated using a Boltzmann equation.

5500ms

Tetracaine

0,024h4h

1h0h

Cultivation at 32°C

After 2 days of cell incubation at 37°C cell were kept at 32°C forvarying length of time -> 24h, 4h, 1h, 0h.
All cells were subsequently measured side by side on one chip (4x).
-> Current amplitudes are significantly higher (~2.4-fold) withovernight incubation (24 hrs) at 32°C.

The IC50 matches theliterature value of 356µM8exactly.
90% Success Rate
QCs: R >100MΩ, I > 0.2nA (4x)completed experiments.
✓ Current-voltage relationships and values for Vhalf of activation for hNaV1.7 and 1.8 calculated using automatedpatch clamp agree well with the literature.
✓ IC50 values for tetracaine and lidocaine were as expected. Tetracaine exhibited state dependence on NaV1.7 and1.8.
✓ Stem cell-derived neurons were used on the automated patch clamp device, the Patchliner, where TTX-sensitive
Nav-mediated currents were recorded. In current clamp mode, action potentials were abolished by lidocaine.

✓ Acknowledgements: special thank you to Matthew Fuller at Icagen for contributing the Nav1.9 data, and toSébastien Nicolas and Michel de Waard at L’insitut du thorax and Rémy Béroud from Smartox for contributing theProtoxin-II data.