Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum.
Sheep cardiac muscle sarcoplasmic reticulum ryanodine receptors have been isolated by density-gradient centrifugation following solubilisation with the zwitterionic detergent, CHAPS. The functional state of the receptor complex has been assessed by quantification of [3H]ryanodine binding and by characterisation of single-channel conductance and gating properties following reconstitution into unilamellar proteo-liposomes and incorporation into planar phospholipid bilayers. A method of solubilisation is described which yields a receptor displaying high-affinity [3H]ryanodine binding (Kd 2.8 nM, Bmax 352 pmol/mg protein) and which functions as a cation-selective, ligand-regulated channel under voltage clamp conditions. Previous reports of channel activity of purified rabbit skeletal and canine cardiac muscle ryanodine receptors describe a range of sub- or variable-conductance events. In contrast, the sheep cardiac ryanodine receptor-channels isolated using the optimal conditions described in this report consistently display a single open state conductance with either Ca2+ or K+ as the charge carrying species.
Lindsay
A R
AR
Department of Cardiac Medicine, National Heart and Lung Institute, University of London, U.K.
Williams
A J
AJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002793:Cholic Acids; D004553:Electric Conductivity; D008051:Lipid Bilayers; D008081:Liposomes; D009124:Muscle Proteins / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry*; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000502:physiology*; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D012756:Sheep
D002793:Cholic Acids; D008051:Lipid Bilayers; D008081:Liposomes; D009124:Muscle Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium