Journal of Muscle Research and Cell Motility 15, 86-101 (1994) Characterization study of the ryanodine receptor and of calsequestrin isoforms of mammalian skeletal muscles in relation to fibre types E. DAMIANI and A. MARGRETH” Centro di Studio per la Biologia e la Fisiopatofogia muscolare, c/o Dipartimento di Scienze Biomediche Sperimentali, Uniuersita’ di Padoua,via Trieste 75, 35121 Padova, Italia Received 4 March 1993; revised 10 October 1993; accepted 1 October 1993 Summary We have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca’+ -activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 pM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1-7.3. IC,, values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca’+- release channel blocker, did not differ significantly (range 0.3-1.0 PM). The Ca’+-d ependence curve (range 1 nM-10 mM free Ca”‘) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cistemae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cistemae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Cleveland’s peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound foorm and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of CaZ+- dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca’+-release channels in junctional terminal cistemae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of CaZf -release, depending on its isoform composition. Introduction In vertebrate skeletal muscle fibres the majority of Ca2+ required for contraction is released from the terminal cistemae (TC) of the sarcoplasmic reticulum (SR) in response to depolarization of transverse tubules (TT). Ca2+ release from TC following muscle excitation is mediated by a class of Ca2+channelsthat are characterist- ically sensitive to plant alkaloids caffeine and ryanodine, as well as to drugs like doxorubicin, and whose activity is modulated by Ca’+, MgZf and ATP (see Palade et al., 1989; and Fleischer & Inui, 1989 for review), but not *To whom correspondence should be addressed. 0142-4319 0 1994 Chapman & Hall directly by membrane voltage. The coupling between excitation and contraction involves a membrane charge movement as the first event (Schneider & Chandler, 1973). Current views favour the dihydropiridine receptor (DHP-R) on junctional TT (Block et al., 1988) acting as the voltage sensormolecule that undergoes a conformational change which is transmitted to the ryanodine receptor (RyR)/Ca’+-release channel of TC at junctional triads (Fleischer & Inui, 1989). In spite of considerable advancement in molecular knowledge of excitation-contraction coupling and of SR Ca2+ release (see Rios et al., 1992; and Dulhunty, 1992, for review), a question that has yet to be resolved is whether these mechanisms, although being basically