Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

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Abstract
Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming α1A-subunits of human P/Q-type Ca2+ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human α1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of α1A-WT with each α1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba2+ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of α1A-mutants increased, suggesting the presence of an α1A-mutant-specific suppression effect. When we coexpressed α1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of α1A-WT and mutant using a splice variant of α1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-α1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.

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