Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP(3)Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP(3)Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (K-d) of similar to 4 mu M at physiological voltage (-40 mV) and intracellular Ca2+ concentration ([Ca2+](i); 10 mu M). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+](i) from similar to 20 to 12 mu M, but did not alter the slope or maximal P-o. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP(3)R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP(3)R1(+/+)) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP(3)R1-deficient (IP(3)R1(-/-)) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP(3)R1 is located in close spatial proximity to BKCa alpha subunits. The IP(3)R1 monoclonal antibody coimmunoprecipitated IP(3)R1 and BKCa channel alpha and beta 1 subunits from cerebral arteries. In summary, data indicate that IP(3)R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.