title: Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.
abstract: The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: S L
authors.initials: SL
authors.lastname: Hamilton
authors.forename: J
authors.initials: J
authors.lastname: Codina
authors.forename: M J
authors.initials: MJ
authors.lastname: Hawkes
authors.forename: A
authors.initials: A
authors.lastname: Yatani
authors.forename: T
authors.initials: T
authors.lastname: Sawada
authors.forename: F M
authors.initials: FM
authors.lastname: Strickland
authors.forename: S C
authors.initials: SC
authors.lastname: Froehner
authors.forename: A M
authors.initials: AM
authors.lastname: Spiegel
authors.forename: L
authors.initials: L
authors.lastname: Toro
authors.forename: E
authors.initials: E
authors.lastname: Stefani
mesh_terms: D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008861:Microsomes / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D011233:Precipitin Tests; D011978:Receptors, Nicotinic / Q000302:isolation & purification; D012964:Sodium / Q000502:physiology
chemical_list: D015220:Calcium Channels; D002793:Cholic Acids; D003902:Detergents; D008051:Lipid Bilayers; D011978:Receptors, Nicotinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012964:Sodium; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate