title: Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups.
abstract: The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.
authors.affiliation: Department of Pharmacology, University of Ottawa, Ontario, Canada.
authors.forename: B J
authors.initials: BJ
authors.lastname: Murphy
authors.forename: A W
authors.initials: AW
authors.lastname: Washkurak
authors.forename: B S
authors.initials: BS
authors.lastname: Tuana
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D004095:Dihydropyridines / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009568:Nitrendipine / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D004095:Dihydropyridines; D004220:Disulfides; D013438:Sulfhydryl Compounds; D002731:4-Chloromercuribenzenesulfonate; D009568:Nitrendipine; D011494:Protein Kinases; D005978:Glutathione; D004229:Dithiothreitol; D007460:Iodoacetamide