Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy.
Bottom Line: The gating pore current, also called omega current, consists of a cation leak through the typically nonconductive voltage-sensor domain (VSD) of voltage-gated ion channels.Two Na(v)1.5 mutations (R222Q and R225W) located in the VSD are associated with atypical clinical phenotypes involving complex arrhythmias and dilated cardiomyopathy.Using the patch-clamp technique, in silico mutagenesis, and molecular dynamic simulations, we tested the hypothesis that these two mutations may generate gating pore currents, potentially accounting for their clinical phenotypes.
Affiliation: Centre de Recherche de L'Institut Universitaire en Santé Mentale de Québec, Québec City, Québec G1J 2G3, Canada.Show MeSH
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Mentions: The nature of the R to Q or W mutation and their spatial localization on the Nav1.5 protein (Fig. 1, R2, R3, respectively; S4/DI) prompted us to examine the possible existence of a gating pore current through the mutant channels. We recorded nonleak-subtracted currents induced by 80-ms voltage steps from −100 to 40 mV. Fig. 4 shows examples of raw data of WT and mutant channel gating pore currents recorded in Cs+ solutions. The corresponding offline linear leak subtraction showed that a nonlinear component appears on the mutant channels but not on the WT channel (Fig. 4 A, bottom traces). After an offline subtraction of the linear leak, the gating pore currents were normalized to the cell capacitance to obtain the gating pore current density–voltage relationships for the WT and mutant channels (Fig. 4 B). These gating pore currents were also recorded in the presence of 10 µM TTX to completely block the central pore (Fig. 4 B). The resulting gating pore currents were indistinguishable from traces without TTX, demonstrating that the current does not flow through the usual α pore of the protein. Fig. 4 C shows the normalized voltage dependence of gating pores based on the theoretical reversal potential of Cs+ in the recordings solutions. The R222Q and R225W mutations show a V1/2 of 1.2 ± 3.7 mV (n = 7) and 13.5 ± 5.7 mV (n = 7), and a slope factor of −19.0 ± 1.7 mV (n = 7) and −15.1 ± 2.2 mV (n = 7), respectively. The gating pore current density at 40 mV was larger for the R222Q mutant (6.6 ± 1.0 pA/pF, n = 8, without TTX and 6.7 ± 0.6 pA/pF, n = 7, with TTX) than for the WT channel (0.17 ± 0.09 pA/pF, n = 5) and R225W mutant (2.7 ± 0.3 pA/pF, n = 10, without TTX and 2.4 ± 0.4 pA/pF, n = 6, with TTX) channels (Fig. 4 D). The α current at −10-mV and the gating pore current at 40-mV relationship is shown Fig. 4 E. A linear correlation was found (r2 = 0.85 for R222Q channels and r2 = 0.86 for R225W channels), indicating that the amplitude of gating pore currents is correlated to the amplitude of the α pore current. The gating pore created by the mutations allowed the passage of K+, Cs+, and Na+ ions while it excludes the passage of NMDG+ (Fig. 5). Correction for the electrochemical gradient yielded the relative permeation for the ions. The gating pores of the two mutations were less permeable to K+ and Na+ than to Cs+, whereas no permeation was found with NMDG+ (Fig. 5). Indeed, the relative permeability of the mutant channels was approximately two times higher for Cs+ than for K+ (2.2 ± 0.3 for the R222Q channel and 2.0 ± 0.2 for the R225W channel) (Fig. 5 C), whereas the relative permeability of Na+ was lower than for K+ (0.59 ± 0.08 for the R222Q channel and 0.67 ± 0.05 for the R225W channel; Fig. 5 C).
Affiliation: Centre de Recherche de L'Institut Universitaire en Santé Mentale de Québec, Québec City, Québec G1J 2G3, Canada.