Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2.
Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).
Nakao
S
S
Department of Biochemistry, Jichi Medical School, Tochigi, Japan.
Ebata
H
H
Hamamoto
T
T
Kagawa
Y
Y
Hirata
H
H
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D066298:In Vitro Techniques; D008564:Membrane Potentials; D008978:Mollusk Venoms / Q000494:pharmacology; D009206:Myocardium / Q000032:analysis*; D011510:Proteolipids; D012508:Sarcolemma / Q000032:analysis; D020960:omega-Conotoxins
D000634:Aminoquinolines; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D008978:Mollusk Venoms; D011510:Proteolipids; D020960:omega-Conotoxins; C020485:proteoliposomes; C053423:Conus magus toxin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C035738:Quin2-acetoxymethyl ester; D002118:Calcium