Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.
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.
Hamilton
S L
SL
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Codina
J
J
Hawkes
M J
MJ
Yatani
A
A
Sawada
T
T
Strickland
F M
FM
Froehner
S C
SC
Spiegel
A M
AM
Toro
L
L
Stefani
E
E
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
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