Reconstitution of the skeletal muscle ryanodine receptor-Ca2+ release channel protein complex into proteoliposomes.
The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized 30 S ryanodine receptor (RyR)/Ca2+ release channel complex from rabbit skeletal muscles, purified by density gradient centrifugation, was reconstituted with an excess of phospholipid into proteoliposomes by removal of the detergent by dialysis. Reconstituted proteoliposomes were concentrated by centrifugation, frozen and thawed, and sonicated. [3H]Ryanodine binding measurements indicated close to 50% recovery of calculated binding activity following reconstitution of the purified RyR and dynamic light scattering measurements a mean vesicle diameter of approximately 150 nm. Using these values, a functional RyR was estimated to be present in only a small fraction (< 15%) of the reconstituted vesicles. SDS-polyacrylamide gel electrophoresis of trypsin-treated proteoliposomes revealed that about four-fifths of the reconstituted 30 S complex was readily accessible to proteolytic attack. Vesicle-45Ca2+ flux and fusion of proteoliposomes with planar lipid bilayers showed that the reconstituted channel complex could be activated by Ca2+ and ATP, inhibited by Mg2+ and ruthenium red, and modified by ryanodine, similarly as observed for native sarcoplasmic reticulum vesicles. These results suggest that the method described here results in the reconstitution of a functional channel and thus provides the opportunity to study the structure and function of the sarcoplasmic reticulum Ca2+ release channel under well defined conditions.
Lee
H B
HB
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Xu
L
L
Meissner
G
G
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism*; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D014357:Trypsin
D015220:Calcium Channels; D002793:Cholic Acids; D003902:Detergents; D009124:Muscle Proteins; D011510:Proteolipids; D019837:Ryanodine Receptor Calcium Release Channel; C020485:proteoliposomes; D014357:Trypsin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium