1. Two mutant human skeletal muscle voltage-gated Na+ channel alpha-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 lines (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 approximately equal to 0.2 mM) and decreased for T1313M (IC50 approximately equal to 1.3 mM) compared with wild-type (IC50 approximately 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 (Kd = 63 microM) but little changed for R1448C (Kd = 14 microM) compared with wild-type (Kd = 11 microM). 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.