Abstract
Large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene are ubiquitously expressed, and they play a role in regulation of many cell types. In excitable cells, BKCa channels and voltage-activated Ca2+ channels often form functional complexes that allow the cytoplasmic domains of BKCa channels to lie within spatially discrete calcium microdomains. Here, we report a novel protein interaction between the β1-subunit of L-type voltage-activated calcium channels (Cavβ1) and critical regulatory domains of Slo1 that can occur in the absence of other proteins. This interaction was identified by a yeast two-hybrid screen, and it was confirmed by confocal microscopy in native neurons, by coimmunoprecipitation, and by direct binding assays. The Cavβ1 subunit binds within the calcium bowl domain of Slo1 that mediates a portion of high-affinity Ca2+ binding to BKCa channels and also to a noncanonical Src homology 3 (SH3) domain-binding motif within Slo1. Binding of Cavβ1 markedly slows Slo1 activation kinetics, and it causes a significant decrease in Ca2+ sensitivity in inside-out and in dialyzed cells, even in the absence of pore-forming subunits of voltage-gated Ca2+ channels. The guanylate kinase domain of Cavβ1 mediates Slo1 regulation through its binding to calcium bowl domains, and this domain of Cavβ1 is necessary and sufficient for the observed effects on BKCa activation. Binding of Cavβ1 to SH3-binding motifs may stabilize the interaction with Slo1, or it may contribute to formation of other complexes, but it does not seem to affect Ca2+-dependent gating of Slo1. Binding of Cavβ1 does not affect cell surface expression of Slo1 in human embryonic kidney 293T cells.
Footnotes
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This study was supported by National Institutes of Health grant NS32748. S.Z. and S.J. contributed equally to this research.
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ABBREVIATIONS: BKCa, large-conductance Ca2+-activated K+;Cavβ1, β1 subunit of voltage-activated Ca2+; Slo1, pore-forming subunit of BKCa channel; Cav, voltage-gated Ca2+; SH3, Src homology 3; GK guanylate kinase; HA, hemagglutinin; FITC, fluorescein isothiocyanate; E9, embryonic day 9; CG, ciliary ganglion; GST glutathione transferase; PBST, phosphate-buffered saline containing 0.2% Triton X-100; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RFP, red fluorescent protein; ANOVA, analysis of variance.
- Received August 8, 2007.
- Accepted November 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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