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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Relative resistance to slow inactivation of human cardiac Na⁺ channel hNa_v1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6

Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana

Submitted 23 August 2007 ; accepted in final form 4 October 2007

Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated Na⁺ channels (Na_vs). To further study its role and understand differences between SI phenotypes of different Na_v isoforms, we analyzed several domain 2-segment 6 (D2-S6) mutants of the human cardiac hNa_v1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole cell Na⁺ currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild type, whereas recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNa_v1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to that of V930K. Therefore, K or Q substitution eliminates hNa_v1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 shows no enhancement of SI, and in fact, V930A and V930C, as well as L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus V930 in D2-S6 appears to be an important structural determinant of SI gating in hNa_v1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Na_vs, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.

sodium channel; voltage gating; site-directed mutagenesis

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