ROLE OF AN S4-S5 LINKER IN SODIUM-CHANNEL INACTIVATION PROBED BY MUTAGENESIS AND A PEPTIDE BLOCKER

A pair of conserved methionine residues, located on the cytoplasmic li nker between segments S4 and S5 in the fourth domain of human heart Na channels (hH1), plays a role in the kinetics and voltage dependence o f inactivation. Substitution of these residues by either glutamine (M( 1651)M(1652)/QQ) or alanine (MM/AA) increases the inactivation time co nstant (tau(h)) at depolarized voltages, shifts steady-state inactivat ion (h(infinity)) in a depolarized direction, and decreases the time c onstant for recovery from inactivation. The data indicate that the mut ations affect the rate constants for both binding and unbinding of a h ypothetical inactivation particle from its binding site. Cytoplasmic a pplication of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W.A. Catterall. 1994. Neuron. 12: 1041-1048.). KI FMK produces a concentration-dependent, voltage-independent increase i n the decay rate of MM/QQ and MM/AA. currents at positive membrane pot entials (K-i similar to 30 mu M), while producing only a small increas e in the decay rate of wild-type currents at a concentration of 200 mu M. Although MM/QQ inactivates similar to-2.5-fold faster than MM/AA. in the absence of peptide, the estimated rate Constants for peptide bl ock and unblock do not differ in these mutants. External Na+ ions anta gonize the block by cytoplasmic KIFMK of MM/AA channels, but not the i nactivation kinetics of this mutant in the absence of peptide. The eff ect of external [Na+] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channel s when they are open and that peptide block does not mimic the inactiv ation process.