Voltage Sensor Module in NaV1.4: S1-S3 Charges Regulate Fast Inactivation

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We investigated the role of charged amino acid residues in the extracellular (ENC) and intracellular (INC) clusters of S1-S3 segments for each of four domains in skeletal muscle sodium channel NaV1.4. We used charge reversing and substituting mutations to show that the extracellular cluster of negative charge in domains III and IV promotes fast inactivation and limits its recovery. E1051R (S1) and D1069K (S2) in domain II slowed entry of channels into fast inactivation and accelerated recovery; E1051D and D1069E each rescued wild type fast inactivation parameters. In domain IV, destabilization of the fast-inactivated state by E1373K (S1) was not rescued in the E1373N mutation, whereas charge-dependent destabilization of fast inactivation was observed with mutations at N1389 (S2). Mutations within the ENC of domains I and II produced lesser effects on fast inactivation, but did inhibit activation. Effects on charge reversing mutations within the INC included an increase in the probability of fast inactivation from closed states. Homology models of the voltage sensor module of each domain were constructed from alignment of sequences of hNaV1.4 to NaVAb, using Modeller. Homology models were constructed for S1 through S4-S5 linker regions, basing the models on the data inferred from the crystal structure of the bacterial sodium channel (Payandeh et al., 2011). The models for hNaV1.4 voltage sensor module in domains I to IV support the hypothesis that S1-S3 counter charges influence the movement of S4 during activation and may explain the effects of mutations in these segments to regulate fast inactivation in a mammalian sodium channel.