Stem cell-derived sensory neurons modelling inherited erythromelalgia: normalization of excitability

Abstract

Effective treatment of pain remains an unmet healthcare need that requires new and effective therapeutic approaches. NaV1.7 has been genetically and functionally validated as a mediator of pain. Preclinical studies of NaV1.7-selective blockers have shown limited success and translation to clinical studies has been limited. The degree of NaV1.7 channel blockade necessary to attenuate neuronal excitability and ameliorate pain is an unanswered question important for drug discovery. Here, we utilize dynamic clamp electrophysiology and induced pluripotent stem cell-derived sensory neurons (iPSC-SNs) to answer this question for inherited erythromelalgia, a pain disorder caused by gain-of-function mutations in Nav1.7. We show that dynamic clamp can produce hyperexcitability in iPSC-SNs associated with two different inherited erythromelalgia mutations, NaV1.7-S241T and NaV1.7-I848T. We further show that blockade of approximately 50% of NaV1.7 currents can reverse neuronal hyperexcitability to baseline levels.

Keywords: Inherited erythromelalgia; Nav1.7; dynamic clamp; iPSC; stem cell-derived sensory neurons.

Figure 1

Kinetic models of Na_V1.7 wild-type and mutant channels. To create kinetic models of Na_V1.7 channels, time constants of (A) activation and deactivation and (B) fast-inactivation and recovery from inactivation for Na_V1.7-WT (black open circles), Na_V1.7-S241T (orange open circles), and Na_V1.7-I848T (blue open circles) are derived from voltage-clamp recordings of these channels in HEK293 cells, co-transfected with the human β-subunits. (C) Representative traces of modelled wild-type Na_V1.7 currents in response to graded depolarizations from −120 mV to +35 mV in 5 mV increments. (D) Close-up of the activation phase of currents evoked by 100 ms depolarizations to −25 and −20 mV for the Na_V1.7-WT kinetic model (hashed blue lines) and Na_V1.7-WT stably expressing HEK293 cells transfected with the β1- and β2-subunits (black line). Example activation (E), fast-inactivation (F), and slow-inactivation (G) curves for the Na_V1.7-WT (black open circles), Na_V1.7-S241T (orange open circles), and Na_V1.7-I848T (blue open circles) illustrate voltage-dependent currents with biophysical properties appropriate for Na_V1.7 channels.

Figure 2

Quantification of Na_V1.7 current density in control iPSC-SNs. (A) Scatter plot depicting the Na_V1.7 current density (−759.10 ± 89.53 pA/pF, n = 15) in iPSC-SNs. (B) Representative traces from one iPSC-SN illustrating sodium currents and Na_V1.7 current quantification in iPSC-SNs. Na_V1.7 current was calculated as the difference between the baseline total sodium current density (black) and the PF-05089771-resistant sodium current density (hashed line). To confirm that high doses of PF-05089771 did not block non-Na_V1.7 currents, tetrodotoxin application resulted in a significant reduction in sodium currents (red).

Figure 3

IEM mutations in Na_V1.7 confer hyperexcitability to iPSC-SNs. (A) Representative traces depicting the same iPSC-SN at baseline (left, black), when 50% of the wild-type Na_V1.7 current is replaced with Na_V1.7-I848T current via dynamic clamp (middle, blue), and when 50% of the wild-type Na_V1.7 current is replaced with Na_V1.7-S241T current via dynamic clamp (right, orange). (B) Replacing 50% of the wild-type Na_V1.7 current with Na_V1.7-S241T current reduced current threshold to 81.09 ± 2.33% of baseline (Student’s paired t-test P < 0.0001, n = 16). (C) Replacing 50% of the wild-type Na_V1.7 current with Na_V1.7-S241T current increased repetitive action potential firing in response to a 1000 ms current injection twice the amplitude of the current threshold by 83.70 ± 12.12% (Student’s paired t-test P < 0.0001, n = 16). (D) Similarly, the Na_V1.7-I848T mutation reduced current threshold to 91.48 ± 1.80% of baseline (Student’s paired t-test P = 0.0004, n = 16). (E) Replacement of half the wild-type Na_V1.7 current with the Na_V1.7-I848T current increased action potential repetitive firing by 58.73 ± 11.36% (Student’s paired t-test P < 0.0001, n = 14).

Figure 4

Hyperexcitability in Na_V1.7-S241T iPSC-SNs can be reversed by dynamic clamp reduction in Na_V1.7 currents. (A) Representative traces from an example iPSC-SN depicting the determination of current threshold with graded 200 ms current injections at baseline (black, 65 pA), Na_V1.7-S241T (orange, top right, 50 pA), and with various degrees of Na_V1.7 current subtraction via dynamic clamp. Normalization of current threshold (defined as current threshold returning to baseline levels) was reached with subtraction of 80% of the total Na_V1.7 current. (B) Representative traces of repetitive action potential firing with a 1000 ms duration current injection at twice the threshold current in the same iPSC-SN as Fig. 4A. Illustrated are baseline repetitive firing levels in a control iPSC-SN with only wild-type Na_V1.7 (black, eight action potentials generated), Na_V1.7-S241T repetitive firing (orange, centre top row, 18 action potentials generated), and traces depicting various degrees of Na_V1.7 current subtraction and their respective action potentials fired. In this neuron, subtraction of 80% of the Na_V1.7 current normalized repetitive firing (i.e. reduced repetitive firing to no more than equal to baseline levels).

Figure 5

Hyperexcitability in Na_V1.7-I848T iPSC-SNs can be reversed by dynamic clamp reduction in Na_V1.7 currents. (A) Representative traces from one iPSC-SN depicting the determination of current threshold with graded 200 ms current injections at baseline (black, 55 pA), Na_V1.7-I848T (blue, top right, 50 pA), and with various degrees of Na_V1.7 current subtraction via dynamic clamp. Normalization of current threshold (defined as current threshold returning to baseline levels) was reached with subtraction of 40% of the total Na_V1.7 current. (B) Representative traces of repetitive action potential firing with a 1000 ms duration current injection at twice the threshold current in the same iPSC-SN as Fig. 4A. Illustrated are baseline repetitive firing levels (black, nine action potentials generated), Na_V1.7-I848T repetitive firing (blue, top row, 12 action potentials generated), and traces depicting various degrees of Na_V1.7 current subtraction and their respective action potentials fired. In this neuron, subtraction of 60% of the Na_V1.7 current normalized repetitive firing (i.e. reduced repetitive firing to no more than equal to baseline levels).

Figure 6

Hyperexcitability in IEM iPSC-SNs can be reversed by approximately a one-half reduction in Na_V1.7 current. (A) Current threshold can be normalized (i.e. returned to control levels) by subtraction of 55.56 ± 8.01% (n = 9) of Na_V1.7 current in Na_V1.7-S241T iPSC-SNs and 44.29 ± 6.85% (n = 7) of Na_V1.7 current in Na_V1.7-I848T iPSC-SNs. Ordinates show percent block of current needed to restore current threshold and repetitive firing to control levels. (B) Repetitive firing can be corrected by subtraction of 53.33 ± 8.17% and 48.00 ± 8.00% of Na_V1.7 current in Na_V1.7-S241T (n = 9) and Na_V1.7-I848T (n = 5) iPSC-SNs.