Functional reconstitution of skeletal muscle Ca2+ channels: separation of regulatory and channel components.
Regulatory properties of a partially purified Ca2+ -channel preparation from isolated rabbit skeletal muscle triads were examined in proteoliposomes. These properties included (i) inhibition by phenylalkylamine antagonists, such as verapamil, (ii) inhibition by the GTP-binding protein Go in the presence of guanosine 5'-[gamma-thio]triphosphate, and (iii) regulation of phenylalkylamine inhibition as a result of phosphorylation by a polypeptide-dependent protein kinase (PK-P). By selective reconstitution of protein fractions obtained by wheat germ lectin and ion-exchange chromatography, a separation of Ca2+-channel activity (fraction C) from regulatory component(s) (fraction R) responsible for verapamil sensitivity was achieved. Reconstitution of fraction C alone yielded vesicles that exhibited channel-mediated 45Ca2+ uptake that could be directly inhibited by coreconstitution of Go in the presence of guanosine 5'-[gamma-thio]triphosphate. However, the 45Ca2+ uptake obtained with fraction C was not inhibited by verapamil. Coreconstitution of fractions C and R yielded vesicles in which the sensitivity of 45Ca2+ uptake to verapamil was restored. The verapamil sensitivity of this preparation could be inhibited by PK-P. Fraction C, obtained by wheat germ agglutinin-Sepharose chromatography followed by DEAE-Sephacel chromatography, included a 180-kDa protein that was phosphorylated by cAMP-dependent protein kinase (PK-A) but not by PK-P and a 145-kDa protein (180 kDa under nonreducing conditions) that was not phosphorylated by either kinase. Fraction R contained proteins that did not adsorb to wheat germ lectin and included 165-kDa and 55-kDa proteins that were phosphorylated by PK-P but not by PK-A. These results suggest a complex model for Ca2+-channel regulation in skeletal muscle involving a number of distinct, separable protein components.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000502:physiology*; D046911:Macromolecular Substances; D009132:Muscles / Q000032:analysis*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits
D002121:Calcium Channel Blockers; D007473:Ion Channels; D046911:Macromolecular Substances; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium