GTP gammaS removal of D-600 block of skeletal muscle excitation-contraction coupling

G proteins interacting with dihydropyridine receptors (DHPR) in transverse tubules (TT) of skeletal muscle may have a role in skeletal excitation-contraction (EC) coupling. The aim of this study was to determine the effects of G protein-specific nucleotides [guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) and guanosine 5'-O-(2-thiodiphosphate) (GDP betaS)] on the EC coupling mechanism in the presence of D-600, an agent that blocks EC coupling by immobilizing the voltage-sensing subunit of the DHPR in its inactivated state. By use of the mechanically peeled single-fiber preparation from rabbit adductor magnus skeletal muscle, 50 microM GTP gammaS and 500 microM GDP betaS were applied with the fiber in a D-600-induced state of blocked EC coupling. Neither nucleotide served as an independent stimulus for sarcoplasmic reticulum (SR) Ca2+ release when added to the TT polarizing bath under conditions of D-600 block. The presence of GTP gammaS or GDP betaS during a complete EC coupling cycle removed the D-600 block of EC coupling, despite continuous bath D-600. After the nucleotides were washed out, in the continued presence of D-600, the D-600 block of EC coupling was reestablished. In contrast, GTP gammaS added only during the period of TT depolarization under D-600 block did not remove the D-600 block of EC coupling, even though GTP gammaS did stimulate SR Ca2+ release. GTP gammaS had no effect on submaximum (0.5-1.0 mM) caffeine contractures and thus is unlikely to be acting through the Ca2+-induced Ca2+ release mechanism of the SR. These data suggest that the molecular binding site for GTP gammaS and GDP betaS is likely to be in the TT near the DHPR, perhaps on a G protein.