Abstract
Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegic migraine, including missense mutations (T263M, T376M, R383H, A606T, R763H, M829R, R834Q, R937P, and X1021R), a deletion mutant (del(K935-S940)ins(I)), and a frameshift mutation (S966fs). According to the Na(+)/K(+)-ATPase crystal structure, a subset of the mutated residues (Ala(606), Arg(763), Met(829), and Arg(834)) is involved in important interdomain H-bond networks, and the C terminus of the enzyme, which is elongated by the X1021R mutation, has been implicated in voltage dependence and formation of a third Na(+)-binding site. Upon heterologous expression in Xenopus oocytes, the analysis of electrogenic transport properties, Rb(+) uptake, and protein expression revealed pronounced and markedly diverse functional alterations in all ATP1A2 mutants. Abnormalities included a complete loss of function (T376M), impaired plasma membrane expression (del(K935-S940)ins(I) and S966fs), and altered apparent affinities for extracellular cations or reduced enzyme turnover (R383H, A606T, R763H, R834Q, and X1021R). In addition, changes in the voltage dependence of pump currents and the increased rate constants of the voltage jump-induced redistribution between E(1)P and E(2)P states were observed. Thus, mutations that disrupt distinct interdomain H-bond patterns can cause abnormal conformational flexibility and exert long range consequences on apparent cation affinities or voltage dependence. Of interest, the X1021R mutation severely impaired voltage dependence and kinetics of Na(+)-translocating partial reactions, corroborating the critical role of the C terminus of Na(+)/K(+)-ATPase in these processes.
Highlights
Familial hemiplegic migraine (FHM)3 is a rare autosomal dominant variant of migraine with aura
L764P and W887R led to nonfunctional proteins [5, 28], whereas others resulted in partially active enzymes with decreased (T345M) or increased (R689Q and M731T) Kϩ affinities or reduced turnover rates (R689Q and M731T) [29, 30]
T376M and del(K935S940)ins(I) showed no significant Rbϩ uptake (p Ͼ 0.05 compared with uninjected oocytes), and values for T263M, R937P, and S966fs were below 20% of WT (Fig. 2B), confirming drastically reduced ion pumping activity
Summary
Familial hemiplegic migraine (FHM)3 is a rare autosomal dominant variant of migraine with aura. Analysis of plasma membrane fractions revealed strongly reduced levels of mutants S966fs and del(K935-S940)ins(I) (Fig. 2C), indicating that impaired targeting to the cell surface accounts for the absence of pump currents in oocytes expressing these mutants.
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