Disrupted coupling of gating charge displacement to Na+ current activation for DIIS4 mutations in hypokalemic periodic paralysis

Abstract

Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K(+). Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K(+) during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K(+), yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na(+) current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na(+) was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼ 10 µS/cm(2) or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na(+) current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na(+) current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of NaV, CaV, and KV channels associated with human disease.

Figure 1.

Currents, recorded under different conditions for the same oocyte, to measure I_Na, I_g, and I_GP. First, Na⁺ currents were recorded in a 115-mM Na⁺ bath with analogue compensation for linear capacitance and digital P/N subtraction of residual linear leak (top row). Responses are shown for test potentials of −20 to 30 mV. The external solution was then exchanged with K⁺/TTX (R672G) or NMDG⁺/TTX (R669H or WT), and P/N leak subtraction was performed to reveal the nonlinear gating charge displacement current, I_g (middle row). Finally, all analogue and digital P/N leak subtraction was omitted to reveal the gating pore current for mutant channels and the residual leakage current for oocytes expressing WT channels (bottom row). Each column shows the responses recorded from a single oocyte (black, WT; blue, R669H; red, R672G).

Figure 2.

Voltage dependence for peak I_Na, normalized by maximal on-gating charge displacement, Q_{on_max}. Symbols show mean and SEM for WT (black; n = 15), R669H (blue; n = 34), and R672G (red; n = 18). Oocytes for which the maximal I_Na occurred at a voltage of less than −30 mV were excluded to reduce the effect of inadequate voltage-clamp control. Error bars represent ± SEM.

Figure 3.

Coupling of gating charge displacement to peak ionic current is diminished and more variable for HypoPP mutants than for WT channels. (A) Each symbol represents the maximal peak I_Na and associated Q_{on_max} for a separate oocyte. Lines show the best fit constrained through the origin. (B) The ensemble coupling behavior for oocytes among each channel type is summarized in a box plot. For each oocyte, the ratio I_Na/Q_{on_max} was computed. Box plots show the mean (circle), 50% level (bar), 25–75% range (box), and 10–90% range (whiskers). The coupling ratio was lower for HypoPP mutant channels than in WT (P < 0.0001). Error bars represent ± SEM.

Figure 4.

Gating pore currents in HypoPP mutant channels. (A) The gating pore current was measured as the steady-state current at the end of a 150-ms pulse that was leak corrected by subtraction of the average steady-state current recorded from oocytes expressing WT Na_V1.4. The gating pore current from each oocyte was normalized by Q_{on_max} for the same egg and plotted as a function of test potential. Symbols show mean and SEM for R669H mutants (blue; n = 32) and for R672G mutants (red; n = 18). Error bars represent ± SEM. (B) The correlation between the gating pore current measured at −140 mV and Q_{on_max} is shown as a scatter plot. Each symbol represents the response from a separate oocyte. Lines show the best fit constrained through the origin. The correlation for I_GP–Q_{on_max} had much less variance than for I_Na–Q_{on_max} measured from the same set of oocytes (Fig. 3 A).