Abstract

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels encoded by the Slo1 gene are ubiquitously expressed, and they play a role in regulation of many cell types. In excitable cells, BK_Ca channels and voltage-activated Ca²⁺ channels often form functional complexes that allow the cytoplasmic domains of BK_Ca 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 (Ca_vβ1) 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 Ca_vβ1 subunit binds within the calcium bowl domain of Slo1 that mediates a portion of high-affinity Ca²⁺ binding to BK_Ca channels and also to a noncanonical Src homology 3 (SH3) domain-binding motif within Slo1. Binding of Ca_vβ1 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 Ca_vβ1 mediates Slo1 regulation through its binding to calcium bowl domains, and this domain of Ca_vβ1 is necessary and sufficient for the observed effects on BK_Ca activation. Binding of Ca_vβ1 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 Ca_vβ1 does not affect cell surface expression of Slo1 in human embryonic kidney 293T cells.