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The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating.

ABSTRACT: Inactivation of voltage-gated calcium channels is crucial for the spatiotemporal coordination of calcium signals and prevention of toxic calcium buildup. Only one member of the highly conserved family of calcium channel beta-subunits--Ca(V)beta--inhibits inactivation. This unique property has been attributed to short variable regions of the protein; however, here we report that this inhibition actually is conferred by a conserved guanylate kinase (GK) domain and, moreover, that this domain alone recapitulates Ca(V)beta-mediated modulation of channel activation. We expressed and refolded the GK domain of Ca(V)beta(2a), the unique variant that inhibits inactivation, and of Ca(V)beta(1b), an isoform that facilitates it. The refolded domains of both Ca(V)beta variants were found to inhibit inactivation of Ca(V)2.3 channels expressed in Xenopus laevis oocytes. These findings suggest that the GK domain endows calcium channels with a brake restraining voltage-dependent inactivation, and thus facilitation of inactivation by full-length Ca(V)beta requires additional structural determinants to antagonize the GK effect. We found that Ca(V)beta can switch the inactivation phenotype conferred to Ca(V)2.3 from slow to fast after posttranslational modifications during channel biogenesis. Our findings provide a framework within which to understand the modulation of inactivation and a new functional map of Ca(V)beta in which the GK domain regulates channel gating and the other conserved domain (Src homology 3) may couple calcium channels to other signaling pathways.

SUBMITTER: Gonzalez-Gutierrez G

PROVIDER: S-EPMC2544601 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating.

Gonzalez-Gutierrez Giovanni G Miranda-Laferte Erick E Nothmann Doreen D Schmidt Silke S Neely Alan A Hidalgo Patricia P

Proceedings of the National Academy of Sciences of the United States of America 20080905 37

Inactivation of voltage-gated calcium channels is crucial for the spatiotemporal coordination of calcium signals and prevention of toxic calcium buildup. Only one member of the highly conserved family of calcium channel beta-subunits--Ca(V)beta--inhibits inactivation. This unique property has been attributed to short variable regions of the protein; however, here we report that this inhibition actually is conferred by a conserved guanylate kinase (GK) domain and, moreover, that this domain alone ...[more]

PMID: 18776052

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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

The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating.

Publications

The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating.

Similar Datasets

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