A human muscle Na+ channel mutation in the voltage sensor IV/S4 affects channel block by the pentapeptide KIFMK
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...
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Published in | The Journal of physiology Vol. 518; no. 1; pp. 13 - 22 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Oxford, UK
The Physiological Society
01.07.1999
Blackwell Science Ltd Blackwell Science Inc |
Subjects | |
Online Access | Get full text |
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Summary: | 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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ISSN: | 0022-3751 1469-7793 |
DOI: | 10.1111/j.1469-7793.1999.0013r.x |