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Functional analysis of human Na(+)/K(+)-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes.


ABSTRACT: AIM:Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine (FHM2, SHM). METHODS:cRNA of human Na(+)/K(+)-ATPase ?2- and ?1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative ?/? interaction sites or in the ?2-subunit's C-terminal region were investigated. Mutants were analyzed by the two-electrode voltage-clamp (TEVC) technique on Xenopus oocytes. Stationary K(+)-induced Na(+)/K(+) pump currents were measured, and the voltage dependence of apparent K(+) affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na(+) buffers to analyze kinetics and voltage-dependent pre-steady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS:Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant differences in the voltage dependence of K(+)-induced currents, and analysis of the transient currents indicated that the extracellular Na(+) affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K(+) interaction, D999H affected the voltage dependence of K(+)-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na(+) was reduced. CONCLUSION:The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.

SUBMITTER: Spiller S 

PROVIDER: S-EPMC4050117 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Functional analysis of human Na(+)/K(+)-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes.

Spiller Susan S   Friedrich Thomas T  

World journal of biological chemistry 20140501 2


<h4>Aim</h4>Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine (FHM2, SHM).<h4>Methods</h4>cRNA of human Na(+)/K(+)-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the two-electrode voltage-clamp (TEVC) technique on Xenopus oocytes. Stationary K(+)-induced Na(+  ...[more]

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