Evidence for direct interaction of G(s)α with the Ca2+ channel of skeletal muscle

S. L. Hamilton, J. Codina, M. J. Hawkes, A. Yatani, T. Sawada, F. M. Strickland, S. C. Froehner, A. M. Spiegel, L. Toro, E. Stefani, L. Birnbaumer, A. M. Brown

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60 Scopus citations


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).

Original languageEnglish (US)
Pages (from-to)19528-19535
Number of pages8
JournalJournal of Biological Chemistry
Issue number29
StatePublished - 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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