title: The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer.
abstract: The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.
authors.affiliation: Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27599.
authors.forename: F A
authors.initials: FA
authors.lastname: Lai
authors.forename: M
authors.initials: M
authors.lastname: Misra
authors.forename: L
authors.initials: L
authors.lastname: Xu
authors.forename: H A
authors.initials: HA
authors.lastname: Smith
authors.forename: G
authors.initials: G
authors.lastname: Meissner
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D005976:Glutaral / Q000494:pharmacology; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D003432:Cross-Linking Reagents; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium; D005976:Glutaral