In this study, we identified a mutation, M1627K, in Nav1.7 from a previously unreported family with PEPD. Life-long symptoms in two members of the family are controlled by the sodium channel blocker carbamazepine. We investigated the functional effect of the M1627K substitution on Nav1.7, and confirm that it causes a large depolarizing shift in the voltage-dependence of steady-state fast-inactivation, with no effect on channel activation. Using current clamp, we show that M1627K mutant channels lower threshold for single action potentials and increase the number of action potentials in response to graded suprathreshold stimuli in small DRG neurons, most of which are nociceptors. Thus we show, for the first time, that a PEPD mutation produces nociceptor hyperexcitability.
Missense mutations in Nav1.7 have been linked to two types of inherited painful neuropathies, early-onset IEM [12–18], and PEPD . While missense mutations in Nav1.7 have been found in most families with IEM and PEPD in whom the gene was sequenced, both IEM and PEPD may be genetically heterogenous because mutations in the coding exons of SCN9A were not found in 5 cases of PEPD , or in cases of familial early-onset  and adult-onset IEM , suggesting that other target genes or mutations in non-coding regions of SCN9A might underlie these cases. Mutations in the coding exons of sensory neuron-specific sodium channels Nav1.8 and Nav1.9 have been ruled out as causative in these cases of inherited erythromelalgia . We present in this study a previously unreported familial case of PEPD from the UK with the mutation M1627K, in the DIV/S4–S5 linker, which is also present in a sporadic case of a male patient from France [19, 20].
The patients in our study have responded favorably to treatment with carbamazepine, similar to those reported previously . Carbamazepine targets voltage-gated sodium channels  and potently inhibits TTX-S channels in DRG neurons , including those specifically produced by wild-type Nav1.7 channels . Although we did not examine the sensitivity of M1672K channels to carbamazepine in this study, carbamazepine has been shown to block persistent currents generated by I1461T and T1464I PEPD Nav1.7 mutant channels , and it is likely that inhibition of M1672K channel activity contributes to the therapeutic action of carbamazepine in the two patients examined in our study.
Our observations of a 19 mV depolarizing shift in the voltage-dependence of fast-inactivation for M1627K confirm the linkage of impaired fast-inactivation of Nav1.7 and PEPD reported by Fertleman et al . We report here that recovery from fast-inactivation was accelerated in the M1627K, consistent with a destabilized inactivation state of the channel. We have also observed that M1627K display an increased ramp current, similar to IEM mutations [13, 16, 17, 21–25]. M1627K displayed a trend toward a small (< 3 mV) hyperpolarizing shift in activation, but this was not statistically significant. Depolarizing shifts in fast-inactivation have also been observed in some [13, 16, 17] but not all [21–25] mutations that cause IEM. However, the depolarizing shifts in fast-inactivation associated to date with IEM (= 10 mV) are smaller than the shifts in PEPD mutations ( and this study). It is intriguing that the IEM mutation A863P with a +10 mV depolarizing shift in fast-inactivation [13, 16, 17] does not yield symptoms of PEPD, suggesting that other factors, perhaps genetic makeup or bigger shifts in the voltage-dependence of fast-inactivation may contribute to clinical manifestations of the disease.
Gating properties of Nav1.7, for example slow recovery from fast-inactivation and an ability to respond to ramp stimuli [28, 35], suggest that, normally, it may act as a "threshold" channel which boosts subthreshold stimuli, and thus sets the gain in nociceptors [36, 37]. Therefore, it is not surprising that mutations lowering the voltage-threshold for channel activation as in IEM lead to DRG neuron firing in response to a weaker stimulus that may normally be innocuous. Mutations that impair fast-inactivation as in PEPD allow more current to pass through the mutant channel, and thus induce stronger depolarization that brings the DRG neuron closer to the voltage-threshold for all-or-none action potential firing. By analogy to mutations in the cardiac channel Nav1.5 which cause arrhythmias and in neuronal channel Nav1.1 which causes epilepsy , the M1627K PEPD mutation which impairs fast-inactivation of Nav1.7 would be expected to increase repetitive firing, leading to hyperexcitablity of DRG neurons.
Indeed, we now show in this study that a PEPD mutation in human Nav1.7 channels in DRG neurons renders these cells hyperexcitable. Current clamp recordings showed a lower threshold for single action potentials, and an increased firing rate in response to suprathreshold stimuli, but did not show a change in resting membrane potential for DRG neurons expressing M1627K channels. However, Harty et al [13, 16, 17] have shown that depolarization of RMP produced by an IEM mutation (A863P) contributes to, but is not solely responsible for, the increase in DRG neuron hyperexcitability produced by that mutation. Thus, impaired fast-inactivation, accelerated repriming, and the enhanced response to slow depolarizations may all have contributed to the hyperexcitability of DRG neurons expressing M1627K.
The DIV/S4–5 linker is highly conserved in length and sequence (Figure 1C) among all sodium channels described to date, suggesting an important role in the normal functioning of the channel. Increasing the length of DIV/S4–S5 linker in Nav1.4 channels renders the mutant channels non-functional . Importantly, mutations in this linker in several channels underlie pathological conditions [19, 38–44]. Interestingly, substitution of the first methionine (Ma) in this linker (Figure 1C) with a positively charged residue (M1627K) in Nav1.7 causes PEPD ( and this study), while substitution with a hydrophobic residue isoleucine (M1476I) in Nav1.4 causes cold-induced myotonia . Subsitution of the second methionine (Mb) with a positively charged residue (M1652R) in Nav1.5 causes LQT-3 syndrome . All three disorders are linked to hyperexcitability of the cell in which they are expressed, irrespective if it is a neuron or a myocyte. The similarity of the outcome suggests a common mechanism of action, consistent with a conserved function of this linker in channel gating.
Site-directed mutagenesis studies have suggested that the DIV/S4–5 linker contributes to the receptor for the fast-inactivation tripeptide IFM in loop 3 (L3) which links DIII and DIV [45–47]. Structural studies have shown that this linker can acquire an α-helical structure  with several residues including the MaMb (Figure 1C) forming a hydrophobic cluster that is important for inactivation, but indicate that these residues do not interact directly with the IFM motif [47, 49, 50]. Taken together, these studies suggest a model of two antiparallel α-helices ; this structure positions MaMb to interact with Y1470Y1471 (numbers according to Nav1.7) in L3. Substitution of the residues that correspond to Ma ( and this study) or Mb  or Y1470Y1471 residues destabilizes the inactivated state of the channel and yields similar gating changes in several channels. Interestingly, phosphorylation by Fyn kinase of Y1495 which is predicted to interact with Ma in Nav1.5 (equivalent to Y1471 in Nav1.7), produces a significant depolarizing shift in the voltage-dependence but no effect on the rate of steady-state fast-inactivation . Thus the introduction of a charged residue at either of these two sites destabilizes this interaction and leads to impaired binding of the inactivation gate with its receptor.
In summary, our results show that a PEPD mutation produces hyperexcitability in DRG neurons. Our findings also confirm the impairment of fast-inactivation previously associated with PEPD mutations, but show that, in addition, a PEPD mutation can enhance the response of the Nav1.7 channel to small, slow depolarizations and accelerate repriming. These data contribute to a better understanding of the pathophysiology of pain in patients with PEPD and provide additional support for efforts to develop Nav1.7-specific therapeutics for treatment of neuropathic pain.