Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle

Susan L. Hamilton, Juan Codina, Mary J. Hawkes, Atsuko Yatani, Tadao Sawada, Faith M. Strickland, Stanley C. Froehner, Allen M. Spiegel, Ligia Toro, Enrico Stefani, Lutz Birnbaumer, Arthur M. Brown

Research output: Contribution to journalArticle

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Abstract

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 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), α*s but not α*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gsα 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 on subunit of the DHPBP, and, in addition, both αs and the β 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.

Original languageEnglish (US)
Pages (from-to)19528-19535
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number29
StatePublished - Oct 15 1991
Externally publishedYes

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GTP-Binding Proteins
Muscle
Skeletal Muscle
Carrier Proteins
Membranes
Ion Channels
Cell Membrane
Heterotrimeric GTP-Binding Proteins
Wheat Germ Agglutinins
Lipid bilayers
Lipid Bilayers
Second Messenger Systems
Cell membranes
Guanosine Triphosphate
Sedimentation
Detergents
Purification
Sucrose
Proteins
Western Blotting

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hamilton, S. L., Codina, J., Hawkes, M. J., Yatani, A., Sawada, T., Strickland, F. M., ... Brown, A. M. (1991). Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle. Journal of Biological Chemistry, 266(29), 19528-19535.

Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle. / Hamilton, Susan L.; Codina, Juan; Hawkes, Mary J.; Yatani, Atsuko; Sawada, Tadao; Strickland, Faith M.; Froehner, Stanley C.; Spiegel, Allen M.; Toro, Ligia; Stefani, Enrico; Birnbaumer, Lutz; Brown, Arthur M.

In: Journal of Biological Chemistry, Vol. 266, No. 29, 15.10.1991, p. 19528-19535.

Research output: Contribution to journalArticle

Hamilton, SL, Codina, J, Hawkes, MJ, Yatani, A, Sawada, T, Strickland, FM, Froehner, SC, Spiegel, AM, Toro, L, Stefani, E, Birnbaumer, L & Brown, AM 1991, 'Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle', Journal of Biological Chemistry, vol. 266, no. 29, pp. 19528-19535.
Hamilton SL, Codina J, Hawkes MJ, Yatani A, Sawada T, Strickland FM et al. Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle. Journal of Biological Chemistry. 1991 Oct 15;266(29):19528-19535.
Hamilton, Susan L. ; Codina, Juan ; Hawkes, Mary J. ; Yatani, Atsuko ; Sawada, Tadao ; Strickland, Faith M. ; Froehner, Stanley C. ; Spiegel, Allen M. ; Toro, Ligia ; Stefani, Enrico ; Birnbaumer, Lutz ; Brown, Arthur M. / Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 29. pp. 19528-19535.
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T1 - Evidence for direct interaction of Gsα with the Ca2+ channel of skeletal muscle

AU - Hamilton, Susan L.

AU - Codina, Juan

AU - Hawkes, Mary J.

AU - Yatani, Atsuko

AU - Sawada, Tadao

AU - Strickland, Faith M.

AU - Froehner, Stanley C.

AU - Spiegel, Allen M.

AU - Toro, Ligia

AU - Stefani, Enrico

AU - Birnbaumer, Lutz

AU - Brown, Arthur M.

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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 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), α*s but not α*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gsα 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 on subunit of the DHPBP, and, in addition, both αs and the β 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.

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