Abstract
Large-conductance Ca2+-activated K+ (BKCa) channels regulate the physiological properties of many cell types. The gating properties of BKCa channels are Ca2+-, voltage- and stretch-sensitive, and stretch-sensitive gating of these channels requires interactions with actin microfilaments subjacent to the plasma membrane. Moreover, we have previously shown that trafficking of BKCa channels to the plasma membrane is associated with processes that alter cytoskeletal dynamics. Here, we show that the Slo1 subunits of BKCa channels contain a novel cytoplasmic actin-binding domain (ABD) close to the C terminus, considerably downstream from regions of the channel molecule that play a major role in determining channel-gating properties. Binding of actin to the ABD can occur in a binary mixture in the absence of other proteins. Coexpression of a small ABD-green fluorescent protein fusion protein that competes with full-length Slo1 channels for binding to actin markedly suppresses trafficking of full-length Slo1 channels to the plasma membrane. In addition, Slo1 channels containing deletions of the ABD that eliminate actin binding are retained in intracellular pools, and they are not expressed on the cell surface. At least one point mutation within the ABD (L1020A) reduces surface expression of Slo1 channels to approximately 25% of wild type, but it does not cause a marked effect on the gating of point mutant channels that reach the cell surface. These data suggest that Slo1-actin interactions are necessary for normal trafficking of BKCa channels to the plasma membrane and that the mechanisms of this interaction may be different from those that underlie F-actin and stretch-sensitive gating.
Footnotes
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This study was supported in part by National Institutes of Health grant NS32748.
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ABBREVIATIONS: BKCa, large-conductance Ca2+-activated K+; Slo1, pore-forming subunit of BKCa channel; GLUT, glucose transporter; GFP, green fluorescent protein; E9, embryonic day 9; HEK, human embryonic kidney; PBS, phosphate-buffered saline; GST, glutathione transferase; PAGE, polyacrylamide gel electrophoresis; PBST, phosphate-buffered saline with 0.2% Triton X-100; ABD, actin-binding domain.
- Received July 6, 2007.
- Accepted November 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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