Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues (‘gating charges’) in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis5 owing to cation leak in both activated and inactivated states6. Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.
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We thank the beamline staff at the Advanced Light Source (BL8.2.1 and BL8.2.2) for assistance during data collection and J. Li for technical and administrative assistance. This research was supported by National Institutes of Health research grants R01 NS015751 (W.A.C.) and R01 HL112808 (W.A.C. and N.Z.), by the Howard Hughes Medical Institute (N.Z.), and by Canadian Institutes of Health Research grant MOP 130461 (R.P.).
Extended data figures and tables
Extended Data Fig. 1 Sequence alignment of the voltage sensor of NavAb with those of human Nav1.4 homologous domain (D)II, Nav1.4 DIV, Cav1.1 DII and Cav1.1 DIV.
Coloured rectangles represent transmembrane helices. Black arrows indicate residues that form the guanidinium binding site, blue arrows indicate the hydrophobic constriction site and red arrows indicate the conserved intracellular negative cluster.
Extended Data Fig. 2 Superposition of the NavAb(WT) voltage sensor and the Electrophorus electricus (electric eel) Nav1.4 DIV voltage sensor.
a–b, Comparison of the conformations of NavAb(WT) voltage sensor (orange) and EeNav1.4 voltage sensor DIV (PDB code: 5XSY) (grey) in side view and top view, respectively. Arginine sensors and hydrophobic residues in the HCS are labelled and shown with side chains in sticks.
a–b, Voltage sensor structure alignment between NavAb(WT) (grey) and NavAb(R3G) (green) in side view and top view, respectively. c–d, Voltage sensor structure alignment between NavAb(WT) (grey) and NavAb(R2G) (cyan) in side view and top view, respectively. Arginine sensors and hydrophobic residues in the HCS are labelled and shown with side chains in sticks.
a, Different conformations of the R4 rotamer in NavAb(R3G) chain A (green) and chain B (orange). b, Different conformations of the R4 rotamer in the four subunits of NavAb in the slow-inactivated state (PDB code: 4EKW).
a, 2mFo−DFc electron density map (blue mesh) of residues around the methylguanidinium binding site at 1σ. b, Overlay of guanidinium binding site (green) and methylguanidinium binding site (orange). c–d, Simulated annealing map (Fo−Fc) contoured at 3σ for methylguanidinium and guanidinium, respectively.
a, Representative gel-filtration chromatography of NavAb(R3G); highlighted peak fractions were concentrated for crystallization. b, Concentrated sample was visualized on SDS–PAGE by Coomassie blue staining.