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

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming α _1A-subunits of human P/Q-type Ca ²⁺ 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 α _1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of α _1A-WT with each α _1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba ²⁺ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of α _1A- mutants increased, suggesting the presence of an α _1A-mutant- specific suppression effect. When we coexpressed α _1A-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 α _1A-WT and mutant using a splice variant of α _1A-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- α _1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.