Mutation I136V alters electrophysiological properties of the Na(v)1.7 channel in a family with onset of erythromelalgia in the second decade

Abstract

Background: Primary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na+ channel NaV1.7 have been linked with primary erythromelalgia. Recently, a new substitution Na(v)1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9-22 years, with onset at 17 years of age or later in 5 of 7 family members), with relatively slow progression (8-10 years) to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1) of domain I (DI) in contrast to all Na(v)1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III.

Results: In this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents.

Conclusion: The I136V substitution in Na(v)1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of Na(v)1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.

Figure 1

I136V mutation alters voltage-dependent activation and steady-state fast inactivation of Na_V1.7 channels. A, Representative family traces of Na+ currents from voltage-clamped HEK293 cells expressing either wild-type Na_V1.7_R (top) or I136V mutant (bottom) channels. Cells were held at -100 mV, and Na⁺ currents were elicited by step depolarizations from -80 to +60 mV in 5 mV increment every 5 seconds. B, Normalized peak current-voltage relationship for Na_V1.7_R (n = 40) and I136V mutant (n = 43) channels. C, Comparison of the voltage-dependent activation and steady-state fast inactivation of Na_V1.7_R and I136V mutant channels. A hyperpolarizing shift (-5.7 mV) of activation was observed in I136V mutant channels. Steady-state fast inactivation was examined using a series of 500-ms prepulses from -140 mV to -10 mV followed by 40-ms test pulses at -10 mV. I136V mutation did not change the V_1/2,fast but altered the slope factor. D, Activation kinetics, measured as time-to-peak, were similar between Na_V1.7_R and I136V mutant channels. E, Fast inactivation kinetics of Na_V1.7_R and I136V mutant channels. Inactivation time constants were calculated by fitting the decay phases of currents shown in Figure 1A with single-exponential function. Gray circles represent I136V shifted 5.7 mV in a depolarizing direction to match the voltage-dependent activation of wild type Na_V1.7_R channels. The inactivation kinetics of I136V channels are slower than that of Na_V1.7_R channels. F, Expanded view of overlap of activation and inactivation Boltzmann fits (area as predicted window current) from C (shaded area for I136V mutation, and lined area for Na_V1.7_R).

Figure 2

I136V mutation alters deactivation and steady-state slow-inactivation. A, Time constants of channel deactivation. Cells were held at -100 mV and tail currents were generated by a brief 0.5-ms depolarization to -20 mV followed by a series of repolarizations ranging from -100 to -40 mV to elicit tail currents (insert a). I136 mutant channels (n = 19) show significantly slower deactivation than Na_V1.7_R channels (n = 17) at all testing potentials. Insert b shows the representative tail currents from two HEK293 cells expressing Na_V1.7_R (solid line) or I136V mutant channels (dotted line). B, I136V mutation significantly enhances steady-state slow-inactivation. Steady-state slow-inactivation was examined by a series of prepulses (30 s) from -130 to +10 mV followed by 100-ms return pulse to -120 mV, then a 20-ms test pulse to -10 mV.

Figure 3

I136V mutation alters recovery from fast-inactivation and increases currents elicited by slow ramp depolarizations. A, Cells were held at -100 mV, and fast-inactivation was initiated by a 20-ms depolarization to 0 mV, followed by a recovery period (2–300 ms) at a recovery potential, followed by a 10-ms test pulse to 0 mV to measure the available channels. Recovery rate were calculated by comparing the peak currents of test pulse to prepulse at 0 mV (20 ms) after various recovery durations (2–300 ms) at different recovery potentials (-100, -90, -80, and -70 mV), and plotted as a function of recovery potentials. Recovery time constants of Na_V1.7_R and I136V mutant channels were then estimated using single-exponential fits. At a recovery potential of -70 mV, I136V mutant channels (n = 11) recovered faster than wild type Na_V1.7_R channels (n = 12). B, Representative ramp currents from Na_V1.7_R (black) and I136V mutant (gray) channels. HEK293 cells were held at -100 mV and a depolarizing voltage ramp from -100 mV to +20 mV was applied at a rate of 0.2 mV/ms. The insert shows mean ramp currents of wild type Na_V1.7_R and I136V mutant channels. Currents were normalized to the maximal peak currents from step depolarizations in Figure 1B. I136V mutant channels were activated at more negative potentials and generated larger ramp currents than Na_V1.7_R channels (Na_V1.7_R: 0.23 ± 0.02, n = 19; I136V: 0.79 ± 0.04, n = 26, p < 0.05).