A204E mutation in Nav1.4 DIS3 exerts gain- and loss-of-function effects that lead to periodic paralysis combining hyper- with hypo-kalaemic signs

Abstract

Periodic paralyses (PP) are characterized by episodic muscle weakness and are classified into the distinct hyperkalaemic (hyperPP) and hypokalaemic (hypoPP) forms. The dominantly-inherited form of hyperPP is caused by overactivity of Na_v1.4 - the skeletal muscle voltage-gated sodium channel. Familial hypoPP results from a leaking gating pore current induced by dominant mutations in Na_v1.4 or Ca_v1.1, the skeletal muscle voltage-gated calcium channel. Here, we report an individual with clinical signs of hyperPP and hypokalaemic episodes of muscle paralysis who was heterozygous for the novel p.Ala204Glu (A204E) substitution located in one region of Na_v1.4 poor in disease-related variations. A204E induced a significant decrease of sodium current density, increased the window current, enhanced fast and slow inactivation of Na_v1.4, and did not cause gating pore current in functional analyses. Interestingly, the negative impact of A204E on Na_v1.4 activation was strengthened in low concentration of extracellular K⁺. Our data prove the existence of a phenotype combining signs of hyperPP and hypoPP due to dominant Na_v1.4 mutations. The hyperPP component would result from gain-of-function effects on Na_v1.4 and the hypokalemic episodes of paralysis from loss-of-function effects strengthened by low K⁺. Our data argue for a non-negligible role of Na_v1.4 loss-of-function in familial hypoPP.

Figure 1

Schema of the pore-forming α subunit of Na_v1.4 and location of A204E. (A) The pore-forming α subunit of hNa_v1.4 is composed of 1.836 amino acid residues forming 4 homologous domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1–S6). The S1–S4 segments of each domain form the voltage-sensor domain, with the positively-charged S4 segments acting as voltage-sensors while the S5 and S6 segments form the selective α pore. More than 70 mutations have been described in human Na_v1.4. Only the ones located in DIS3 or phenotypically-related to A204E are listed on the schema: p.M203K located in DIS3 and associated to a congenital myopathy phenotype; the hypoPP missense mutations substituting positively-charged (+) residues in S4 segments; p.R1451L resulting in a PP phenotype combining hyper- and hypo-PP. The p.R1129Q and p.R1451L are associated with two distinct phenotypes: hypoPP or normoPP (R1129Q), and paramyotonia congenita with hyperPP or PP combining hyper- and hypo-PP signs (R1451L). Hyper PP = hyperkalaemic periodic paralysis; hypoPP = hypokalaemic periodic paralysis; PMC = paramyotonia congenita; NormoPP = normokalaemic PP; CM = congenital myopathy. (B) Model of voltage-sensor domain I based on Na_vAb crystal structure showing the position of the Ala204 residue on the extracellular side of S3. (C) Alignment of the hNa_v1.4 amino acid sequence around the A204 residue with eukaryotic EeNav1.4 (electric eel) and Na_VPaS (putative Na_v channel from the American cockroach) channels whose cryo-electron microscopy structure was determined with a high-level resolution. The A204 residue is not conserved in these orthologs except in EeNav1.4b.

Figure 2

Biophysical properties of wild-type (WT) and mutant A204E hNa_v1.4 channels in HEK293 cells. (A) Representative whole-cell current traces recorded from HEK293 cells expressing wild-type (WT, black) or A204E (red) hNa_v1.4 channels in response to a test pulse of 10 ms at −10 mV from a holding potential of −120 mV are shown. (B) Normalized current/voltage relationships of WT (n = 36) and A204E (n = 28) channels. Current densities were normalized by cell capacitance. (C) Voltage dependence of activation and steady-state fast inactivation curves for WT and A204E channels were plotted and fitted with a single Boltzmann equation. (D) Effect of A204E on window current. The probability of being within this window was calculated as indicated in the materials and methods section. (E) Steady-state fast inactivation for WT and A204E channels in response to the pulse protocol shown in the inset. (F) Recovery from fast inactivation of WT and A204E channels. (G) Voltage-dependence of time constants (τ) of fast inactivation for WT (n = 9) and A204E (n = 9) channels obtained when measuring entry from −70 to −30 mV and recovery from −120 to −80 mV. The statistical differences between WT and A204E channels are shown (***P < 0.001).

Figure 3

Analyses of slow inactivation of wild-type (WT) and mutant (A204E) hNa_v1.4 channels in HEK293 cells. (A) The voltage dependence of entry into slow inactivation was measured using a triple-pulse protocol. The cells were first depolarized at −10 mV for 5 ms, then for 60 sec from a holding potential at −120 mV to +20 mV. A 5 ms test pulse at −10 mV was applied to measure the Na⁺ current after an interval of 20 ms at −120 mV to allow recovery from fast inactivation. (B) The entry into slow inactivation was also measured with a triple-pulse protocol. The resulting curves were fitted to a monoexponential equation. (C) Recovery from slow inactivation. The resulting curves were fitted with a monoexponential equation.

Figure 4

Analyses of gating pore current in oocytes. (A) Representative current traces recorded in response to stimulation protocols from oocytes expressing WT (top, in black), A204E (middle, in red) or R222G (bottom, in green) rNa_v1.4 channels. The dashed line in black represents the zero current. (B) Normalized current-voltage relationships (I-V) for WT, A204E and R222G rNa_v1.4 channels.

Figure 5

Biophysical properties of WT and mutant hNa_v1.4 channels in an extracellular concentration of K⁺ equal to 1mM (1 mM K⁺_ext) compared to 4 mM K⁺ _ext. (A) Voltage-dependence of steady-state activation. (B) Voltage-dependence of steady-state fast inactivation. (C) Voltage-dependence of slow inactivation. (D) Entry into fast inactivation. (E) Time constants of entry into fast inactivation at −60 mV. The statistical differences between WT and A204E values at 4 mM K⁺_ext and 1 mM K⁺ _ext channels are shown (**P < 0.01; ***P < 0.001). (F) Recovery from fast inactivation obtained with a holding potential of −100 mV.