Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli

Abstract

Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM.

Significance statement: Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients.

Keywords: chronic pain; man on fire syndrome; sensory neurons; temperature responses; thermosensation; voltage-gated sodium channel.

Figure 1.

Ala1632Gly family pedigree and structural modeling of Ala1632Gly mutation in the Nav1.7 channel. A, Circles represent females. Squares represent males. Arrow indicates the proband. Filled symbols represent subjects with IEM. +, subjects tested positive for the Ala1632Gly mutation; −, subjects tested negative for the Ala1632Gly mutation. Slash indicates one deceased subject. Age of onset (years) of consenting subjects: I.2: preteen; II.1: ∼10; II.3: ∼5; II.4: ∼10; II.5: ∼7; III.4: ∼7; III.5: not sure; III.9: ∼8. B, Schematic of Nav1.7 channel topology showing the Ala1632Gly mutation. Nav1.7 consists of four homologous domains (DI–DIV), joined by three loops (L1–L3). Sequence alignment shows the alignment of Nav1.1-Nav1.9-containing Ala1632. The equivalent residue is conserved in all human voltage-gated sodium channels, except for Nav1.9, in which the analogous residue is a serine (Ser1496). C, Cytosolic view of the structural model of Nav1.7 channel transmembrane domains. Ala1632 is located in domain IV, at the hinge between S4–S5 linker and the S5 helix. Red represents Ala1632. D, E, Nav1.7 domain III multi-state structural models. Blue represents Nav1.7 WT model. Black represents Ala1632 residue. Yellow represents Nav1.7-A1632G model. Red represents Gly1632 residues. The models are superimposed. D, Nav1.7 WT and Ala1632Gly models in open state. The region containing Ala1632 or Gly1632 is boxed and enlarged on the right side. E, Nav1.7 WT and Ala1632Gly models in closed state. The region containing Ala1632 or Gly1632 is boxed and enlarged on the right side. The position and orientation of Gly1632 have a radial tuning compared with Ala1632.

Figure 2.

Biophysical properties of Nav1.7-A1632G mutant channels. A, B, Representative current traces recorded from HEK293 cells expressing WT (A) and Ala1632Gly mutant (B) channels. C, Voltage dependence of activation of WT and Ala1632Gly was plotted and fitted with a single Boltzmann equation. D, Voltage dependence of steady-state fast inactivation of WT and Ala1632Gly was plotted and fitted with a single Boltzmann equation.

Figure 3.

Ala1632Gly increases the number of spontaneously firing small DRG neurons and reduces current threshold for evoked firing. A, Representative recording of a spontaneously firing small DRG neuron expressing Ala1632Gly. The trace is a record of activity over 30 s without current injection. Bar graph represents a 3.2-fold increase in the proportion of spontaneously firing DRG neurons expressing Ala1632Gly (orange) compared with WT hNav1.7 channels (blue). B, Current threshold was significantly reduced in neurons expressing Ala1632Gly channels. C, Responses of a current-clamped small DRG neuron transfected with WT hNav1.7 channels to a series of subthreshold (180–190 pA) and suprathreshold depolarizing current steps (195 and 200 pA). Starting at a subthreshold stimulus, the current amplitude was increased in 5 pA increments to stimulus strength above threshold. The current threshold was 195 pA for this neuron. D, The same threshold protocol was applied to a small DRG neuron transfected with hNav1.7-A1632G channel. The current threshold was 105 pA for this neuron. Arrows indicate the current amplitude used to elicit the labeled response. Inset, Superimposed action potential waveforms of WT (blue) and Ala1632Gly expressing neurons (orange). Error bars indicate SEM. *p < 0.05.

Figure 4.

The Ala1632Gly mutation increases evoked firing frequency in small DRG neurons. Responses of a small DRG neuron expressing WT (A–C) or Ala1632Gly (D–F) hNav1.7 channels to 500 ms depolarizing current steps that are onefold (A, D), twofold (B, E), and threefold (C, F) of its threshold, respectively. G, Summary of firing frequency in response to graded inputs. Error bars indicate SEM. *p < 0.05. H, Resting membrane potential of neurons expressing Ala1632Gly displays a mild depolarization of 1.8 mV compared with neurons expressing WT, but this depolarization does not reach statistical significance (p = 0.189).

Figure 5.

Increased temperature enhances firing of DRG neurons expressing hNav1.7-A1632G mutant channels. Current-clamp recordings of DRG neurons at different temperatures. A, B, Percentages of spontaneously firing DRG neurons expressing Ala1632Gly (A, left panel) or WT (A, right panel) at 33°C, and Ala1632Gly (B, left panel) or WT (B, right panel) at 40°C. C, Responses of DRG neurons to depolarizing current steps at a set holding membrane potential (V_m) = −60 mV at 33°C (C, left panel), and 40°C (C, right panel). *p < 0.05 (repeated-measures two-way ANOVA with Bonferroni corrections; n = 8–10).

Figure 6.

MEA recording of spontaneous firing of DRG neurons expressing WT or Ala1632Gly mutant channels. A, B, Representative spike waveforms recorded using MEA from DRG neurons expressing WT Nav1.7 channels (A) and Nav1.7-A1632G mutant channels (B). Black line indicates the averaged waveform from 8 individual spikes. Dark cyan lines indicate each individual spike. C, D, Raw traces from a single electrode recording DRG neurons expressing WT Nav1.7 channels (C) and Nav1.7-A1632G mutant channels (D). DRG neurons expressing Nav1.7-A1632G mutant channels produce more spikes compared with those expressing WT Nav1.7 in the same recording period. E, F, Well-wide (64 electrodes) raster plot of MEA recordings of DRG neurons expressing WT (E) and hNav1.7-A1632G (F) at 37°C. Each horizontal plot represents recording from one electrode. More electrodes recorded spikes, and at higher frequencies from DRG neurons expressing Nav1.7-A1632G mutant channels. G–N, Heat maps of representative MEA recordings from DRG neurons expressing WT or hNav1.7-A1632G mutant channel. The firing frequency of each active electrode is color-coded: White/red represents high firing frequency. Blue/black represents low firing frequency. Each circle represents an active electrode within an 8 × 8 electrode array. For DRG neurons expressing Nav1.7-A1732G mutant channels (G–J), three active electrodes were evident at 33°C (G), four at 37°C (H), five at 40°C (I), and six at 45°C (J). Neurons expressing hNav1.7-A1632G mutant channel fired at a relatively high firing frequency. For DRG neurons expressing WT channels (K–N), three active electrodes were evident at 33°C (K), 37°C (L), and 40°C (M). Four active electrodes were seen at 45°C (N). These neurons fired at a relatively low frequency. O, Average firing frequencies of neurons expressing Nav1.7-A1632G and WT channel at the four temperatures (33°C, 37°C, 40°C, and 45°C). Forty active electrodes recorded firing from DRG neurons expressing WT channels (pooled from a total of 8 rats), whereas 162 active electrodes recorded firing from DRG neurons expressing Ala1632Gly mutant channels (pooled from a total of 8 rats). *p < 0.05 (pairwise Mann–Whitney test with FDR corrections). **p < 0.01 (pairwise Mann–Whitney test with FDR corrections). P, Average numbers of active electrodes from 3 wells containing DRG neurons expressing Nav1.7-A1632G and WT channels at the four temperatures. *p < 0.05 (repeated-measures two-way ANOVA with Bonferroni corrections). **p < 0.01 (repeated-measures two-way ANOVA with Bonferroni corrections). ***p < 0.01 (repeated-measures two-way ANOVA with Bonferroni corrections).