A human muscle Na+ channel mutation in the voltage sensor IV/S4 affects channel block by the pentapeptide KIFMK

1. Whole cell patch clamping of transfected HEK293 cells was used to examin e the effects of a pentapeptide (KIFMK) containing the proposed inactivatio n particle of the Na+ channel on two mutations causing myotonia. One mutati on (R1448P) is located in the voltage sensor IV/S4, and the other one (G130 6E) near the postulated inactivation gate within the III-IV linker. 2. In the absence of peptide, currents or wild-type (WT) and mutant human m uscle Na+ channels decayed monoexponentially with inactivation time constan ts that were 5-fold (R1448P) and S-fold (G1306E) larger for the mutants. Up on intracellular application of KIFMK (0.3-1 mM) tl-le current decay became biexponential with an additional fast decaying component that increased in amplitude with depolarization. 3. Furthermore, the peptide induced large tail currents upon repolarization , indicating that KIFMK prevents inactivation by blocking open Na+ channels . The peak of this tail current decreased only slowly with depolarizations of increasing duration. The voltage dependence of this decline indicated th at the dissociation rate of the charged peptide decreased with depolarizati on. Increased external [Na+] ([Na+](e)) antagonized block by KIFMK, consist ent with a pore-blocking mechanism. 4. The results are discussed with regard to a three-state model for one ope n, an absorbing inactivated and one blocked state with voltage-dependent on - and off-rates for peptide binding. The peptide had qualitatively similar effects on WT and both mutants, indicating that the freely diffusible pepti de accelerates the current decay in all three clones. However, for the R144 8P mutation the affinity for KFIMK was decreased and the voltage dependence of peptide block was changed in a similar way to the voltage dependence of inactivation. These data suggest that the mutation R1448P affects the volt age-dependent formation of a receptor site for both the inactivation partic le and KIFMK.