title: Characterization study of the ryanodine receptor and of calsequestrin isoforms of mammalian skeletal muscles in relation to fibre types.
abstract: 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(2+)-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 microM 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. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca(2+)-release channel blocker, did not differ significantly (range 0.3-1.0 microM). The Ca(2+)-dependence curve (range 1 nM-10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae 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 form 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 Ca(2+)-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(2+)-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca(2+)-release, depending on its isoform composition.
authors.affiliation: Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Italia.
authors.forename: E
authors.initials: E
authors.lastname: Damiani
authors.forename: A
authors.initials: A
authors.lastname: Margreth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D002155:Calsequestrin / Q000032:analysis* / Q000302:isolation & purification; D004317:Doxorubicin / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D009132:Muscles / Q000737:chemistry* / Q000502:physiology; D009200:Myocardial Contraction; D009206:Myocardium / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel
chemical_list: D015220:Calcium Channels; D002155:Calsequestrin; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D004317:Doxorubicin; D002118:Calcium