Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca(2+) concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca(2+) concentration (Ca(2+) sparks) to repolarizing spontaneous transient outward K(+) currents (STOCs). BK channels are composed of channel-forming BKalpha and auxiliary BKbeta1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca(2+). To assess the in vivo functions of this ss subunit, mice with a disrupted BKbeta1 gene were generated. Cerebral artery VSMCs from BKbeta1 -/- mice generated Ca(2+) sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKbeta1 -/- mice have an abnormal Ca(2+) spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKbeta1 -/- mice responded to agonist and elevated KCl with a increased contractility. BKbeta1 -/- mice had higher systemic blood pressure than BKbeta1 +/+ mice but responded normally to alpha(1)-adrenergic vasoconstriction and nitric oxide-mediated vasodilation. We propose that the elevated blood pressure in BKbeta1 -/- mice serves to normalize Ca(2+) spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

Abstract-Ca2ϩ sparks are localized intracellular Ca 2ϩ events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca 2ϩ spark triggering depends on precise delivery of Ca 2ϩ ions through dihydropyridine (DHP)-sensitive Ca 2ϩ channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca 2ϩ influx channels and RyRs. Because caveolae contain DHP-sensitive Ca 2ϩ channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca 2ϩ sparks. Using methyl-␤-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca 2ϩ sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca 2ϩ sparks were not significantly affected. We ruled out the possibility that the decreases in Ca 2ϩ spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca 2ϩ load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca 2ϩ sparks in a caveolae microdomain. The transient elevation in [Ca 2ϩ ] i at the inner mouth of a single caveolemmal Ca 2ϩ channel induces simultaneous activation and thus opens several RyRs to generate a local Ca 2ϩ release event, a Ca 2ϩ spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca 2ϩ signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease. Materials and MethodsSingle SMCs were isolated enzymatically from myogenic cerebral (100 to 800 m in diameter posterior and basilar) arteries from adult Sprague-Dawley rats (12 to 14 weeks; 200 to 280 g), as previously described. 14 Single cardiomyocytes were isolated enzymatically from newborn rats. 21 For Ca 2ϩ imaging, the cells were incubated with the Ca 2ϩ indicator fluo-3-AM (5 m) and pluronic acid (0.005% wt/vol) for 30 minutes at room temperature in Ca 2ϩ -free Hanks solution. 3,14 SMCs and cardiomyocytes were imaged using a BioRad laser scanning confocal microscope attached to a Nikon Diaphot microscope. Whole-cell membrane currents and potentials in freshly isolated cerebral artery myocytes were measured using the perforated patch configuration of the patch-clamp technique configuration with amphotericin B or nystatin. 22 Currents were recorded from holding potentials of Ϫ80 mV (Ϫ100 mV) during lineage voltage ramps at 0.67 V/s from Ϫ100 to ϩ100 mV or 300-ms step pulses to different potentials; pulse frequency 0.2 Hz. 22,23 An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org. ResultsWe used a laser scanning confocal microscope and the Ca Figure 1 online, available at http:// www.circresaha.org) by membrane depolarization (using 60 mmol/L external K ϩ ) or by th...

Abstract-Intracellular