TY - JOUR
T1 - Evidence for direct interaction of G(s)α with the Ca2+ channel of skeletal muscle
AU - Hamilton, S. L.
AU - Codina, J.
AU - Hawkes, M. J.
AU - Yatani, A.
AU - Sawada, T.
AU - Strickland, F. M.
AU - Froehner, S. C.
AU - Spiegel, A. M.
AU - Toro, L.
AU - Stefani, E.
AU - Birnbaumer, L.
AU - Brown, A. M.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1991
Y1 - 1991
N2 - The α 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 α subunit of G(s) 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), α(s)* but not α(i-3)* stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between G(s)α 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 α(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 α(s)* but not α(i-3)*. G proteins were immunoprecipitated with an antibody to the α1 subunit of the DHPBP, and, in addition, both α(s) and the β subunit of G(s) were detected in Western blots of the partially purified DHPBP. The results suggest that G(s) and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for G(s).
AB - The α 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 α subunit of G(s) 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), α(s)* but not α(i-3)* stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between G(s)α 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 α(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 α(s)* but not α(i-3)*. G proteins were immunoprecipitated with an antibody to the α1 subunit of the DHPBP, and, in addition, both α(s) and the β subunit of G(s) were detected in Western blots of the partially purified DHPBP. The results suggest that G(s) and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for G(s).
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M3 - Article
C2 - 1655789
AN - SCOPUS:0025937759
VL - 266
SP - 19528
EP - 19535
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -