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Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

ABSTRACT: Ca2+ channel beta subunits modify alpha1 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 beta4 subunit had alpha1 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 beta4b 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 approximately 30% made possible the building of beta4a and beta4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta4b 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 beta4a versus beta4b in the human CNS. Whereas beta4a is distributed throughout evolutionarily older regions of the CNS, beta4b is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta4 subunit has played in the evolution of complex neural networks.

SUBMITTER: Helton TD

PROVIDER: S-EPMC6758066 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

Helton Thomas D TD Kojetin Douglas J DJ Cavanagh John J Horne William A WA

The Journal of neuroscience : the official journal of the Society for Neuroscience 20021101 21

Ca2+ channel beta subunits modify alpha1 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 beta4 subunit had alpha1 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 s ...[more]

PMID: 12417658

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Project description:High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca(V)2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation.

Dataset Information

Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

Publications

Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

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