title: Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: evidence for low-affinity sites and for the involvement of G proteins.
abstract: Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites (Kd = 0.30 +/- 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 microM for the agonist (+/-)-Bay K8644 and for the antagonists nifedipine, (+/-)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 microM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) on binding parameters. At a concentration of 10 microM, neither GTP gamma S nor GDP beta S significantly affected the binding of dihydropyridines to their high-affinity sites. GTP gamma S did, however, increase the ability of (+/-)-Bay K8644, but not of (+/-)-nitrendipine, to accelerate the rate of dissociation of tightly bound (+)-[3H]PN200-110. GDP beta S did not affect the dose dependence of either the agonist or the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
authors.forename: S M
authors.initials: SM
authors.lastname: Dunn
authors.forename: C
authors.initials: C
authors.lastname: Bladen
mesh_terms: D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D017275:Isradipine; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D009568:Nitrendipine / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D013237:Stereoisomerism; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D008024:Ligands; D008565:Membrane Proteins; D010069:Oxadiazoles; D011978:Receptors, Nicotinic; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); C038806:1,4-dihydropyridine; D009568:Nitrendipine; D019204:GTP-Binding Proteins; D017275:Isradipine