Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells

Intracellular Ca2+ levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca2+-dependent relaxation is mediated by discretely localized Ca2+ release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca2+ concentration, termed sparks, stimulate nearby Ca2+-activated K+ (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca2+ spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca2+ sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca2+ sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca2+. sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F-o), width at half-maximal amplitude, and duration compared with wild-type cell Ca2+ sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca2+ sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 mu mol/L), external Ca2+ removal, and depletion of SR Ca2+ stores by caffeine (1 mmol/L), Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca2+ spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.