title: Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.
abstract: The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: C
authors.initials: C
authors.lastname: Callaway
authors.forename: A
authors.initials: A
authors.lastname: Seryshev
authors.forename: J P
authors.initials: JP
authors.lastname: Wang
authors.forename: K J
authors.initials: KJ
authors.lastname: Slavik
authors.forename: D H
authors.initials: DH
authors.lastname: Needleman
authors.forename: C
authors.initials: C
authors.lastname: Cantu
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.forename: T
authors.initials: T
authors.lastname: Jayaraman
authors.forename: A R
authors.initials: AR
authors.lastname: Marks
authors.forename: S L
authors.initials: SL
authors.lastname: Hamilton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism* / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014316:Tritium; D014357:Trypsin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D008051:Lipid Bilayers; D010446:Peptide Fragments; D014316:Tritium; D012433:Ryanodine; D014357:Trypsin