title: Functional behaviour of the ryanodine receptor/Ca(2+)-release channel in vesiculated derivatives of the junctional membrane of terminal cisternae of rabbit fast muscle sarcoplasmic reticulum.
abstract: We have devised a novel procedure, employing Chaps rather than Triton [Costello B., Chadwick C., Saito A., Chu A., Maurer A., Fleischer S. J Cell Biol 1986; 103: 741-753], for obtaining vesiculated derivatives of the junctional face membrane (JFM) domain of isolated terminal cisternae (TC) from fast skeletal muscle of the rabbit. Enriched JFM is minimally contaminated with junctional transverse tubules. The characteristic ultrastructural features and the most essential features of TC function relating to this membrane domain-i.e. both the Ca(2+)-release system and the Ca2+ and calmodulin (CaM)-dependent protein kinase (CaM I PK) system-appear to be retained in enriched JFM. We show that our isolation procedure, yielding up to a 2.5-fold enrichment in ryanodine receptor (RyR) protein and in the maximum number of high affinity [3H]-ryanodine binding sites, does not alter the assembly for integral proteins associated with the receptor in its native membrane environment, i.e. FKBP-12, triadin and the structurally related protein junction [Jones L.R., Zhang L., Sanborn K., Jorgensen A., Kelley J. J Biol Chem 1995; 270: 30787-30796] having, in common, the property to bind calsequestrin (CS) in overlays in the presence of EGTA. The substrate specificity of endogenous CaM I PK is also the same as that of parent TC vesicles. Phosphorylation of mainly triadin and of a high M(r) polypeptide, and not of the RyR, is the most remarkable common property. Retention of peripheral proteins, like CS and histidine-rich Ca(2+)-binding protein, although not that endogenous CaM, and of a unique set of CaM-binding proteins, unlike that of junctional SR-specific integral proteins, is shown to be influenced by the concentration of Ca2+ during incubation of TC vesicles with Chaps. Characterization of RyR functional behaviour with [3H]-ryanodine has indicated extensive similarities between the enriched JFM and parent TC vessicles, as far as the characteristic bell shaped Ca(2+)-dependence of [3H]-ryanodine binding and the dose-dependent sensitization to Ca2+ by caffeine, reflecting the inherent properties of SR Ca(2+)-release channel, as well as concerning the stimulation of [3H]-ryanodine binding by increasing concentrations of KCl. Stabilizing the RyR in a maximally active state by optimizing concentrations of KCl (1 M), at also optimal concentrations of Ca2+ (pCa 4), rendered the receptor less sensitive to inhibition by 1 microM CaM, to a greater extent in the case of enriched JFM. That was not accounted for by any significant difference in the IC50 concentrations of CaM varying between 40 nM to approximately 80 nM, at low-intermediate and at high KCl concentrations, respectively. Additional results with enriched JFM using doxorubicin, a pharmacological Ca2+ channel allosteric modifier, strengthen the hypothesis that the conformational state at which RyR is stabilized, according to the experimental assay conditions for [3H]-ryanodine binding, directly influences CaM-sensitivity.
authors.affiliation: Department of Experimental Biomedical Sciences, University of Padova, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Damiani
authors.forename: G
authors.initials: G
authors.lastname: Tobaldin
authors.forename: E
authors.initials: E
authors.lastname: Bortoloso
authors.forename: A
authors.initials: A
authors.lastname: Margreth
mesh_terms: D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002793:Cholic Acids; D004076:Digoxigenin; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D015151:Immunoblotting; D007074:Immunoglobulin G; D007425:Intracellular Membranes / Q000378:metabolism; D017275:Isradipine / Q000378:metabolism; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin
chemical_list: D002121:Calcium Channel Blockers; D002147:Calmodulin; D002793:Cholic Acids; D007106:Immune Sera; D007074:Immunoglobulin G; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D004076:Digoxigenin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D017275:Isradipine