1. Two mutant human skeletal muscle voltage-gated Na+ channel α-subunits (hSkM1), with mutations found in patients with hereditary paramyotonia congenita (T1313M on the III-IV linker and R1448C on the outside of S4 of repeat IV), and wild-type hSkM1 channels were expressed in a human embryonic kidney cell line (tsA201) using recombinant cDNA. 2. Compared with wild-type, both mutants exhibited altered inactivation phenotypes. Current decay was slowed for both, but voltage-dependent availability from inactivation was shifted in the negative direction for R1448C and in the positive direction for T1313M. 3. The hypothesis that a local anaesthetic, lidocaine (lignocaine), binds primarily to the inactivated state to block the channel was reassessed by testing lidocaine block of these two mutants and the wild-type channel. 4. T1313M showed reduced phasic block, but R1448C showed increased phasic block for trains of depolarizations. 5. Rest block (from -120 mV) was increased for R1448C (IC50≃ 0.2 mM) and decreased for T1313M (IC50≃ 1.3 mM) compared with wild-type (IC50≃ 0.5 mM), but these differences were diminished at a holding potential of -150 mV, suggesting that the differences were caused by binding to the inactivated state rather than a different affinity of lidocaine for the resting state. 6. Inactivated state affinity measured from lidocaine-induced shifts in voltage-dependent availability was reduced for T1313M (K(d) = 63 μM) but little changed for R1448C (K(d) = 14 μM) compared with wild-type (K(d) = 11 μM). Two pulse recovery protocols showed faster recovery from lidocaine block for T1313M and slower recovery for R1448C. Together these accounted for the opposite effects on lidocaine phasic block observed for the mutant channels. 7. Neither mutation is located at a putative lidocaine binding site in domain 4 S6, yet both affected lidocaine block. The data suggest that R1448C altered phasic lidocaine block mainly through altered kinetics, but T1313M altered block through a change in affinity for the inactivated state. These findings have implications for drug therapy of paramyotonia congenita, and also provide an insight into structural requirements for drug affinity.
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