High-throughput electrophysiology reveals subtype specific temperature dependence of sodium channel
activation and inactivation
Sophia Kriegeskorte1, Raya Bott1, Martin Hampl3, Ralf Hausmann2, Angelika Lampert1
1 University Hospital, RWTH Aachen University, Institute of Physiology, Aachen, North Rhine-Westphalia, Germany 2 University Hospital, RWTH Aachen University, Institute of Clinical Pharmacology, Aachen, North Rhine-Westphalia, Germany 3 Nanion Technologies GmbH, Munich, Germany-30
0.0-80Inactivation V1/2

Activation V1/2✱✱✱✱ns✱✱✱✱

nsnsns✱✱✱✱nsnsns✱✱✱✱✱✱✱35°C10 ms10 ms

Persistent Current
0.2

Nav1.5

15°C

0.2

35°C

0.1

0.0-80

-60

-40

-20

0

I(pers)/I(max)

25°C

15°C
25°C
35°C

0.1

0.0-80

20-60

-40

-20

0

Nav1.60.2

I(pers)/I(max)Nav1.325°C

0.1

0.0-80

20-60

-40

-20

01 containing 140mM NaCl, 10mM Hepes, 5mM Glucose, 4mM KCl, 2mM CaCl2, 1mM MgCl2
2 containing 80mM NaCl, 60mM NMDG, 10mM Hepes, 5mM Glucose, 4mM KCl, 2mM CaCl2, 1mM MgCl23 containing 10mM EGTA, 10mM Hepes, 10mM CsCl, 10mM NaCl, 10mM CsF

0.010

35°C

0.005
0.000-4015°C
25°C

0.010

35°C

0.005

0.000-20

Voltage (mV)

0-40-20

Voltage (mV)

010100

10100

25°C
35°C

0.0
11000

25°C

0.5
Nav1.7WT
Nav1.7 L823R
Nav1.7 I1461T

0.0

1000

110100100

1000

Interpulse interval (ms)Interpulse interval (ms)1.01035°C1.0

0.5
Nav1.7WT
Nav1.7 L823R
Nav1.7 I1461T

0.0
11000

101001000

Interpulse interval (ms)Interpulse interval (ms)

10 ms

35°C

500 pA

15°C

Nav1.7 WT0.10.0-60-40-2025°C

0.2

Nav1.7 I1461T

0.015
25°C

0.010

35°C

0.005
0.000-40-20

Voltage (mV)

01.00.8
0.6
0.40.20Nav1.7 WT
Nav1.7 L823R
Nav1.7 I1461T

0.1

0.020-80-60-40-20035°C

0.2

Nav1.7 L823R
Nav1.7 I1461T

0.1

0.0-8020WT
L823R
I1461T

0.010
0.005
0.000-40-20

Voltage (mV)

00.01525°C

WTL823R

0.010

I1461T

0.005
0.000-40-20

Voltage (mV)

-40-20020

Voltage (mV)

Voltage (mV)

15°C-60035°C
0.015

WTL823R

0.010

5

1.0

0.6
0.4
0.2

Nav1.7WT
Nav1.7 L823R
Nav1.7 I1461T

0.8
0.6
0.4
0.2
0.0

1.4

2.5

5Ramp (mV/ms)

1.4

2.5

-20Voltage (mV)

5Ramp (mV/ms)-The Nav voltage dependence of activation is temperaturedependent.
-While the voltage dependence of fast inactivation is only slightlyaffected, the inactivation kinetics are strongly modulated bytemperature.
-Temperature changes modify the different Nav channels in a
subtype-specific manner; especially Nav1.3 shows a pronounced
sensitivity towards cold .

I1461T

0.005
0.000
-40

0.8

0.0

2.5

Ramps 35°C

Ramps 25°C

CONCLUSION

10 ms

10 ms

Voltage (mV)

15°C

1.00.0500 pA

Inactivation time constant (τ)
0.015

Ramps 15°C

Ramp (mV/ms)

Persistent Current-80

Nav1.6

Nav1.7 L823R shows strongly enhanced ramp currents

An increase of current was observed at colder temperature and atenhanced ramp speed.

-80n=13n= 813n= 5112n=7n= 013n= 5120n=
49n=
66
n=57τTau300 pA

Nav1.7 L823R

20Time constant (s)DataControl384 2.025°C

Nav1.50.015

0.06-60

Persistent Current

Voltage (mV)Time constant (s)SyncroPatch 384

15°C

Time constant (s)HEK cells harvested for a
SyncroPatch experiment

Time constant (s)

0.015

115°C

ns25°C

Inactivation time constant (τ)
Nav1.3✱✱✱ns

Nav1.7WT
Nav1.7 L823R
Nav1.7 I1461T

1.4

0.215°C

Voltage (mV)

Voltage (mV)

0.0Interpulse interval (ms)Both mutations lead to significantly enhanced persistent currents
Nav1.7 WT has small persistent currents, which are strongly enhanced
in both mutations.
Inactivation time constants accelerate with increasing temperature
and are similar for the three Nav channels, but the current decay ofI1461T at 15°C is significantly slowed down.
Nav1.7 I1461T

10 ms

35°C

1000

0.5

1-100

15°C

500 pA

500 pA

4I(pers)/I(max)τ-40-60

Persistent Current
500 pA-20

I(pers)/I(max)

25°C

Voltage (mV)

-80

n=
9n= 3
102n=
99
n=
9n= 4
128n=
69
n=
71n=
87
n=
73

15°C

Nav1.3-HEK 293 cells stably expressing hNav1.3, hNav1.5, hNav1.6, hNav1.7,hNav1.7 L823R and hNav1.7 I1461T were investigated using theautomated patch-clamp system Syncropatch 384 (NanionTechnologies GmbH, Munich). We performed whole-cell voltage
clamp experiments at 15°C, 25°C and 35°C.
-As extracellular solution we used either “External Standard”1 or“External Standard NMDG”2 and as intracellular solution “Internal CsF110”3.
-All data was analyzed using “DataControl384 2.0” (NanionTechnologies) and “GraphPad Prism Version 9” and is depicted asmean (±SEM). Activation and inactivation curves were fitted using aBoltzmann distribution equation.

25°C

0.5

-40

Persistent current and inactivation time constant suggest a
2 strongly slowed inactivation process at colder temperatures
Especially Nav1.3, but also Nav1.5 and very little Nav1.6 are showing
enhanced persistent currents and a strongly prolonged inactivation timeconstants at 15°C compared to 25°C and 35°C.

I(pers)/I(max)METHODSns✱✱✱✱

100

15°C

1.0✱✱✱

ns✱✱-60

Created with BioRender.com

I1461T  PEPD-30✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱

n=90n=84n=64n=80
n=
77
n=
50
n=
71
n=
36
n=
38

Modified from Körner and Lampert✱✱✱-100-60✱✱ns

Voltage (mV)

Voltage (mV)

Voltage (mV)

-40

✱✱✱✱

-90 -70 -50
Voltage (mV)

Inactivation V1/2

Activation V1/2

-40
-20

0.0
-130 -110

1Data fitted using a Boltzmann distribution equation

ns
ns

20

1015°C

Nav1.6

1.0

Normalized current✱✱✱✱✱✱✱

00.0

Normalized current✱✱✱✱-40-20
Voltage (mV)

35°C

0.5

Interpulse interval (ms)Normalized current✱✱✱✱ns

-60

15°C
25°C
35°C

25°C

0.5

Nav1.7 I1461T

1Data fitted using a Boltzmann distribution equation

Nav1.7L823R IEM

-90 -70 -50
Voltage (mV)

0.5

15°C

1I(pers)/I(max)00.0-130 -110

Nav1.7 L823R
15°C
25°C
35°C

Time constant (s)Nav1.6
15°C
25°C
35°C
20

1.0

0.5

1.0

Normalized current

g/gmax0.5

Inactivation1

1.0

Nav1.5

Normalized current

1.0

Nav1.7 WT
15°C
25°C
35°C

Nav1.3n=13
n= 813n= 5112n=7
n= 013n= 5120
n=49
n=66
n=57

We aimed to study the role of temperature on the gating properties of
Nav1.3, 1.5, 1.6, 1.7 and two Nav1.7 mutations, causing IEM (L823R) or
PEPD (I1461T), to better understand their pathophysiological role andthe temperature effects that may underlie disease-linked neuronal
hyperexcitability.

-40-20
Voltage (mV)

Activation1Time constant (s)

AIM

-60

1.0

I/Imax0.5

0.0-80Inactivation1

Nav1.3
15°C
25°C
35°C
Nav1.5
15°C
25°C
35°C

1.0

g/gmax-Patients suffering from inherited erythromelalgia (IEM) or
paroxysmal extreme pain disorder (PEPD), both pain syndromes
linked to gain-of-function mutations in Nav1.7, are showing specificsensitivities towards temperature changes: IEM pain attacks can betriggered by mild warmth, while cold wind is described as a possible
trigger factor in PEPD.

Activation1

Increasing temperature reduces time-dependence of recovery for all
investigated channels. Recovery of Nav1.6, and especiallyNav1.7 I1461T, seem to be most affected by temperature.
Normalized current

affected by temperature.

-It is known that their gating is temperature sensitive but the effecton single Nav-subtypes is not well investigated yet.

Recovery from fast inactivation

Normalized current

shifts activation of Nav1.3, 1.5 and 1.6 to more depolarized
1 Coldpotentials, while steady-state fast inactivation seems only mildly

5

Normalized current-Voltage-gated sodium channels (Navs) play an essential role in theelectrical signaling of cells, especially in action potential generation.

Nav1.7 WT, Nav 1.7 L823R and Nav1.7 I1461T are showing a
3 cold induced shift of activation to more depolarized potentials
L823R vs. WT : Activation is shifted to more hyperpolarized potentials.
I1461T vs. WT: Activation and inactivation are shifted to more
depolarized potentials.
Inactivation is unaltered for WT, while both mutations are showing smalltemperature induced shifts.

Normalized current

RESULTS

I/ImaxBACKGROUND

0-Compared to Nav1.7 WT, both mutations show a pronounced
sensitivity towards temperature changes which may explain theclinical phenotypes of IEM and PEPD.