Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 126, Issue 4, Pages 365-377Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200509354
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Funding
- NCRR NIH HHS [S10 RR 16859] Funding Source: Medline
- NHLBI NIH HHS [R01 HL069911, R01 HL 69911] Funding Source: Medline
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Auxiliary Ca2+ channel beta subunits (Ca-V beta) regulate cellular Ca2+ signaling by trafficking pore-forming alpha(1) subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca-V beta SH3-GK module regulates multiple Ca2+ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca-V beta SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca-V beta effects on channel trafficking, activation gating, and increased open probability (P-o). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca-V beta trafficking and gating properties: alpha(1C) targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P-o functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit. The results reveal that Ca-V beta SH3 and GK domains function codependently to tune Ca2+ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca2+ channel activity.
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