Characterization and cADP-Ribose Activation of Reconstituted Ryanodine Receptors from Coronary Arterial Smooth Muscle
Recent studies indicate that cyclic ADP-ribose (cADPR) serves as a second messenger to mobilize Ca2+ from sarcoplasmic reticulum (SR) independent of IP3. However, the mechanism by which cADPR activates Ca2+ mobilization is poorly understood. In the present study, ryanodine receptors from SR of coronary arterial smooth muscle cells (CASMCs) were reconstituted into planar lipid bilayer. A unitary Ca2+ current through this receptor/channel complex was detected at membrane potentials from -40 to +40 mV. Mean slope conductance of this current was 245 ± 4.5 pS in a symmetric cesium methanesulfonate solution. Ryanodine at 0.1-10 μM produced a typical subconductance with increased channel open probability (NPO), and 50 μM ryanodine, however, decreased the NPO of the channels. These ryanodine effects represent a unique feature of this SR Ca2+ release channel confirmed in other tissues. Addition of Ca2+ into bath solution (0.01-10 μM) significantly increased the NPO of these SR Ca2+ channels. cADPR significantly increased the channel NP0 in a concentration-dependent manner. A 9.7-fold increase in the NPO was observed at a concentration of cADPR of 1 μM. This cADPR-induced activation of the SR Ca2+ channels was completely abolished by 50 μM ryanodine. IP3 had no effect on the activity of these reconstituted SR Ca2+ channels even at the highest concentration studied (10 μM). It is concluded that ryanodine receptor/Ca2+ channels are present in CASMCs and can be reconstituted into planar lipid bilayer. cADPR stimulates Ca2+ release through the activation of ryanodine receptors on the SR of CASMCs.