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

W. Peter, N. Mitrovic *, M. Schiebe, F. Lehmann-Horn and H. Lerche *

Whole cell patch clamping of transfected HEK293 cells was used to examine the effects of a pentapeptide (KIFMK) containing the proposed inactivation particle of the Na⁺ channel on two mutations causing myotonia. One mutation (R1448P) is located in the voltage sensor IV/S4, and the other one (G1306E) near the postulated inactivation gate within the III-IV linker.

In the absence of peptide, currents of wild-type (WT) and mutant human muscle Na⁺ channels decayed monoexponentially with inactivation time constants that were 5-fold (R1448P) and 3-fold (G1306E) larger for the mutants. Upon intracellular application of KIFMK (0·3-1 mM) the current decay became biexponential with an additional fast decaying component that increased in amplitude with depolarization.

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 that the dissociation rate of the charged peptide decreased with depolarization. Increased external [Na⁺] ([Na⁺]_e) antagonized block by KIFMK, consistent with a pore-blocking mechanism.

The results are discussed with regard to a three-state model for one open, 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 peptide accelerates the current decay in all three clones. However, for the R1448P 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 voltage-dependent formation of a receptor site for both the inactivation particle and KIFMK.

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