Alternative splicing of β4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels

Abstract

Ca2+ channel β subunits modify α1 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 β4 subunit had α1 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 ω-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 β4b 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 ∼30% made possible the building of β4a and β4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the β4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of β4b 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 β4a versus β4b in the human CNS. Whereas β4a is distributed throughout evolutionarily older regions of the CNS, β4b is concentrated heavily in the forebrain, These results raise interesting questions about the functional role that alternative splicing of the β4 subunit has played in the evolution of complex neural networks.