title: T-tubule depolarization-induced local events in the ryanodine receptor, as monitored with the fluorescent conformational probe incorporated by mediation of peptide A.
abstract: There is a considerable controversy about the postulated role of the Thr(671)-Leu(690) (peptide A) region of the dihydropyridine (DHP) receptor alpha1 II-III loop. Here we report that peptide A introduced the fluorescence probe methyl coumarin acetamido (MCA) in a well defined region of the ryanodine receptor (RyR), A-site, in a specific manner. Depolarization of the T-tubule moiety of the triad induced a rapid increase of the fluorescence intensity of the MCA attached to the A-site. Other RyR agonists, which activate the RyR without mediation of the DHP receptor (e.g. caffeine, polylysine, and peptide A), induced Ca(2+) release without producing such an MCA fluorescence increase. Both magnitudes of the fluorescence change and Ca(2+) release increased with the increase in the degree of T-tubule depolarization. MCA fluorescence increase at the A-site and subsequent sarcoplasmic reticulum Ca(2+) release were blocked by blocking of the DHP receptor-to-RyR communication. These results may be accounted for by two alternative models as follows. (a) Upon T-tubule depolarization a portion of the DHP receptor comes close to the RyR, forming a hydrophobic interface (within such an interface the A-site is located), or (b) T-tubule depolarization may produce a local conformational change in the A-site-containing region of the RyR that is not necessarily within the DHP receptor/RyR junction.
authors.affiliation: Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ikemoto
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009553:Nimodipine / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D003374:Coumarins; D005456:Fluorescent Dyes; D007476:Ionophores; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D009553:Nimodipine; C030123:coumarin; D002118:Calcium