Received for publication August 8, 2007.
Revised November 7, 2007.
Accepted for publication November 7, 2007.

The 1 subunit of L-type voltage-gated Ca²⁺ channels independently binds to and inhibits the gating of large-conductance Ca²⁺-activated K⁺ channels

Abstract

Large conductance Ca²⁺- activated K⁺ channels (BK_Ca channels) encoded by the Slo1 gene are ubiquitously expressed, and 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 1-subunit of L-type voltage-activated calcium channels (Ca_v1) 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 confirmed by confocal microscopy in native neurons, co-immunoprecipitation, and by direct binding assays. The Ca_v1 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 non-canonical Src Homology 3 (SH3) domain-binding motif within Slo1. Binding of Ca_v1 markedly slows Slo1 activation kinetics and causes a significant decrease in Ca²⁺ sensitivity in inside-out and in dialyzed cells, even in the absence of pore-forming subunits of Ca_v channels. The guanylate kinase (GK) domain of Ca_v1 mediates Slo1 regulation through its binding to calcium bowl domains, and this domain of Ca_v1 is necessary and sufficient for the observed effects on BK_Ca activation. Binding of Ca_v1 to SH3-binding motifs may stabilize the interaction with Slo1, or contribute to formation of other complexes, but does not appear to affect Ca²⁺-dependent gating of Slo1. Binding of Ca_v1 does not affect cell surface expression of Slo1 in HEK293T cells.

Key words: Ion channel regulation, Func. analysis receptor/ion channel mutants, Immunocytochemistry, Single channel kinetics, Yeast 2-hybrid

This article has been cited by other articles:

				G. Gonzalez-Gutierrez, E. Miranda-Laferte, D. Nothmann, S. Schmidt, A. Neely, and P. Hidalgo The guanylate kinase domain of the {beta}-subunit of voltage-gated calcium channels suffices to modulate gating PNAS, September 16, 2008; 105(37): 14198 - 14203. [Abstract] [Full Text] [PDF]