Differential effects of homologous S4 mutations in human skeletal muscle sodium channels on deactivation gating from open and inactivated states

Abstract

The outermost charged amino acid of S4 segments in the α subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild‐type (hSkM1‐wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right‐shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time‐to‐peak activation at ‐20 mV was increased for R1448C and for double mutations. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge‐to‐cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1‐wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. Homologous charge mutations of S4 segments produced domain‐specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non‐identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate‐limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co‐operativity.

The Human Skeletal Muscle Na Channel Mutation R669H Associated with Hypokalemic Periodic Paralysis Enhances Slow Inactivation

“…A charge-neutralizing arginine-to-cysteine substitution at the same position in hSkM1 (R669C) caused only a modest depolarizing shift of the G-V relationship of 5-7 mV (Mitrovic et al, 1998;Groome et al, 1999), similar to the small 3 mV rightward shift reported here. In the human cardiac Na channel isoform hH1, mutation of the outermost arginine in IIS4 to histidine caused a similar modest depolarizing shift in the conductance-voltage relationship and no change in the voltage dependence of inactivation (Chen et al, 1996).…”

Ranolazine block of human Nav1.4 sodium channels and paramyotonia congenita mutants

“…Mutation of this positively charged arginine results in the general set of biophysical defects associated with paramyotonia congenita mutants, including slowed whole-cell current inactivation, reduced voltage-dependence of inactivation, enhanced rate of the recovery from inactivation and increased tetrodotoxin (TTX)-sensitive persistent (late) I Na . [7][8][9][10][11][12] These features lead to enhanced Na + entry into the cell causing depolarization of the resting membrane potential and sustained action potential firing which results in hyperexcitability (myotonia) or inexcitability (paralysis). 10,13,14 Current therapies for patients with…”

µO-Conotoxins Inhibit NaVChannels by Interfering with their Voltage Sensors in Domain-2

“…Since the position of the S4 gating charge is biased towards the cytosolic side, the outermost charges may contribute to the initial gating charge transfer across the transmembrane electric field upon membrane depolarization. [32][33][34][35] When the outermost charge of a sensor is removed, e.g., R663C in domain-2, the initial activation of this sensor (outward movement) should be less voltage-dependent. If this initial movement of the voltage sensor already weakens the stability of the MrVIA-channel interaction, neutralization of the outermost charged residue is expected to have a strong impact on the kinetics with which MrVIA is repelled from the channel.…”