FUNCTIONAL-BEHAVIOR OF THE RYANODINE RECEPTOR CA2-RELEASE CHANNEL IN VESICULATED DERIVATIVES OF THE JUNCTIONAL MEMBRANE OF TERMINAL CISTERNAE OF RABBIT FAST MUSCLE SARCOPLASMIC-RETICULUM()

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 cist ernae (TC) from fast skeletal muscle of the rabbit. Enriched JFM is mi nimally contaminated with junctional transverse tubules. The character istic ultrastructural features and the most essential features of TC f unction relating to this membrane domain - i.e. both the Ca2+-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 ou r isolation procedure, yielding up to a 2.5-fold enrichment in ryanodi ne receptor (RyR) protein and in the maximum number of high affinity [ H-3]-ryanodine binding sites, does not alter the assembly for integral proteins associated with the receptor in its native membrane environm ent, i.e. FKBP-12, triadin and the structurally related protein juncti n [Jones L.R., Zhang L., Sanborn K., Jorgensen A., Kelley J. J Biol Ch em 1995; 270: 30787-30796] having, in common, the property to bind cal sequestrin (CS) in overlays in the presence of EGTA. The substrate spe cificity 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 polypept ide, and not of the RyR, is the most remarkable common property. Reten tion of peripheral proteins, like CS and histidine-rich Ca2+-binding p rotein, although not that of endogenous CaM, and of a unique set of Ca M-binding proteins, unlike that of junctional SR-specific integral pro teins, is shown to be influenced by the concentration of Ca2+ during i ncubation of TC vesicles with Chaps. Characterization of RyR functiona l behaviour with [H-3]-ryanodine has indicated extensive similarities between the enriched JFM and parent TC vesicles, as far as the charact eristic bell shaped Ca2+-dependence of [H-3]-ryanodine binding and the dose-dependent sensitization to Ca2+ by caffeine, reflecting the inhe rent properties of SR Ca2+-release channel, as well as concerning the stimulation of [H-3]-ryanodine binding by increasing concentrations of KCI. Stabilizing the RyR in a maximally active state by optimizing co ncentrations of KCl (1 M), at also optimal concentrations of Ca2+ (pCa 4), rendered the receptor less sensitive to inhibition by 1 mu M CaM, to a greater extent in the case of enriched JFM. That was not account ed for by any significant difference in the IC50 concentrations of CaM , varying between 40 nM to approximately 80 nM, at low-intermediate an d at high KCI concentrations, respectively. Additional results with en riched JFM using doxorubicin, a pharmacological Ca2+ channel allosteri c modifier, strengthen the hypothesis that the conformational state at which RyR is stabilized, according to the experimental assay conditio ns for [H-3]-ryanodine binding, directly influences CaM-sensitivity.