ACTIVATION OF SINGLE CARDIAC AND SKELETAL RYANODINE RECEPTOR CHANNELSBY FLASH-PHOTOLYSIS OF CAGED CA2+

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release ch annels isolated from rabbit skeletal and canine cardiac muscle were re constituted in planar lipid bilayers. Single channel activity was meas ured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Csas charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM- nitrophen) in a small volume directly in front of the bilayer. The fre e [Ca2+] in this small volume and in the bulk solution was monitored w ith Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] s timuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa7-->6) was appl ied to both the cardiac and skeletal release channels. The rate of cha nnel activation was determined by fitting a single exponential to ense mble currents generated from at least 50 single channel sweeps. The ti me constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respec tively. This study presents a method for defining the fast Ca2+ regula tion kinetics of single SR Ca2+ release channels and shows that the ac tivation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.