Alternative splicing of a beta(4) subunit proline-rich motif regulates voltage-dependent gating and toxin block of Ca(v)2.1 Ca2+ channels

Abstract

Ca2+ channel beta subunits modify alpha(1) subunit gating properties through direct interactions with intracellular linker domains. In a previous report (Helton and Horne, 2002), we showed that alternative splicing of the beta(4) subunit had alpha(1) subunit subtype-specific effects on Ca2+ channel activation and fast inactivation. We extend these findings in the present report to include effects on slow inactivation and block by the peptide toxin omega-conotoxin (CTx)-MVIIC. N-terminal deletion and site-directed mutagenesis experiments revealed that the effects of alternative splicing on toxin block and all aspects of gating could be attributed to a proline-rich motif found within N-terminal beta(4b) amino acids 10-20. Interestingly, this motif is conserved within the third postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain of the distantly related membrane-associated guanylate kinase homolog, PSD-95. Sequence identity of similar to30% made possible the building of beta(4a) and beta(4b) three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta(4) N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta(4b) to an extended arm structure that flanks what would be the equivalent of a highly modified PSD-95 carboxylate binding loop. Northern blot analysis revealed a markedly different pattern of distribution for beta(4a) versus beta(4b) in the human CNS. Whereas beta(4a) is distributed throughout evolutionarily older regions of the CNS, beta(4b) is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta(4) subunit has played in the evolution of complex neural networks.