Functional expression of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum in COS-1 cells.
A full-length cDNA encoding the ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was transiently expressed in COS-1 cells. Immunoblotting studies showed that the expressed ryanodine receptor and the native ryanodine receptor of rabbit skeletal muscle were indistinguishable in molecular size and immunoreactivity. Scatchard analysis of [3H]ryanodine binding to transfected COS-1 cell microsomes resulted in a Bmax of 0.22 pmol/mg of protein and a Kd of 16.2 nM. Expressed ryanodine receptors were solubilized in CHAPS and were shown to cosediment with native ryanodine receptors in a sucrose density gradient. Thus, the expressed receptor, like the native receptor, is assembled as a large oligomeric complex. Single-channel recordings in planar lipid bilayers were used to investigate the functional properties of the sucrose gradient-purified complex. The expressed ryanodine receptor formed a large conductance channel activated by ATP and Ca2+ and inhibited by Mg2+ and ruthenium red. Ryanodine reduced the conductance and increased the mean open time in a manner consistent with that of native channels. These results demonstrated that functional binding sites for the physiological ligands (Ca2+, Mg2+, and ATP) and pharmacological ligands (ruthenium red and ryanodine) controlling gating of the Ca2+ release channel are encoded in the ryanodine receptor cDNA and are faithfully expressed in COS-1 cells. Ryanodine receptors expressed in COS-1 cells displayed several conductance states > or = 1 nS not present in native channels. Such anomalous conductance states of the expressed channel might be referable to lack of muscle-specific posttranslational processing or to the need for components not present in COS-1 cells, which may be required to stabilize the channel structure.
Banting and Best Department of Medical Research, University of Toronto, C.H. Best Institute, Ontario, Canada.
S R
SR
Chen
D M
DM
Vaughan
J A
JA
Airey
R
R
Coronado
D H
DH
MacLennan
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004553:Electric Conductivity; D015870:Gene Expression; D007150:Immunohistochemistry; D009124:Muscle Proteins / Q000235:genetics* / Q000502:physiology; D009132:Muscles / Q000737:chemistry*; D010957:Plasmids; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D012995:Solubility; D014162:Transfection
D015220:Calcium Channels; D002793:Cholic Acids; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D000255:Adenosine Triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate