2 HUMAN PARAMYOTONIA-CONGENITA MUTATIONS HAVE OPPOSITE EFFECTS ON LIDOCAINE BLOCK OF NA-CELL LINE( CHANNELS EXPRESSED IN A MAMMALIAN)

1. Two mutant human skeletal muscle voltage-gated Na+ channel alpha-su bunits (hSkM1), with mutations found in patients with hereditary param yotonia congenita (T1313M on the III-IV linker and R1448C on the outsi de 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 fur both, but voltage-dependent availability from inactivation was shifted in the negative direction f or R1448C and in the positive direction for T1313M. 3. The hypothesis that a local anaesthetic, lidocaine (lignocaine), binds primarily to t he inactivated state to block the channel was reassessed by testing li docaine block of these two mutants and the wild-type channel. 4. T1313 M showed reduced phasic block, but R1448C showed increased phasic bloc k for trains of depolarizations 5. Rest block (from -120 mV) was incre ased for R1448C (IC50 approximate to 0.2 mM) and decreased for T1313M (IC50 approximate to 1.3 mM) compared with wild-type (IC50 approximate to 0.5 mM), but these differences were diminished at a holding potent ial of -150 mV, suggesting that the differences were caused by binding to the inactivated state rather than a different affinity of lidocain e for the resting state. 6. Inactivated state affinity measured from l idocaine-induced shifts in voltage-dependent availability was reduced for T1313M (K-d=63 mu M) but little changed for R1448C (K-d = 14 mu M) compared with wild-type (K-d = 11 mu M). Two pulse recovery protocols showed faster recovery from lidocaine block for T1313M and slower rec overy for R1448C. Together these accounted for the opposite effects on lidocaine phasic block observed for the mutant channels. 7. Neither m utation is located at a putative lidocaine binding site in domain 4 S6 , Set both affected lidocaine block. The data suggest that R1448C alte red 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 con genita, and also provide an insight into structural requirements for d rug affinity.