Dominant-negative suppression of Cav2.1 currents by alpha(1)2.1 truncations requires the conserved interaction domain for beta subunits.
Episodic ataxia type 2 (EA2) is an autosomal dominant disorder arising from CACNA1A mutations, which commonly predict heterozygous expression of Ca(v)2.1 calcium channels with truncated alpha(1)2.1 pore subunits. We hypothesized that alpha(1)2.1 truncations in EA2 exert dominant-negative effects on the function of wild-type subunits. Wild-type and truncated alpha(1)2.1 subunits with fluorescent protein tags were transiently co-expressed in cells stably expressing Ca(v) auxiliary beta subunits, which facilitate alpha1 subunit functional expression through high-affinity interactions with the alpha interaction domain (AID). Co-expression of wild-type subunits with truncations often resulted in severely reduced whole-cell currents compared to expression of wild-type subunits alone. Cellular image analyses revealed that current suppression was not due to reduced wild-type expression levels. Instead, the current suppression depended on truncations terminating distal to the AID. Moreover, only AID-bearing alpha(1)2.1 proteins co-immunoprecipitated with Ca(v) beta subunits. These results indicate that Ca(v) beta subunits may play a prominent role in EA2 disease pathogenesis.
Raike
Robert S
RS
Department of Neurology, University of Minnesota, Minneapolis, MN 55455, USA.
Kordasiewicz
Holly B
HB
Thompson
Randall M
RM
Gomez
Christopher M
CM
D000704:Analysis of Variance; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation / Q000379:methods; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology / Q000528:radiation effects; D009154:Mutation / Q000502:physiology*; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014162:Transfection
D020864:Calcium Channels, N-Type; D049452:Green Fluorescent Proteins; D021122:Protein Subunits