Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Aug;85(16):5814–5818. doi: 10.1073/pnas.85.16.5814

Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.

D E Logothetis 1, D H Kim 1, J K Northup 1, E J Neer 1, D E Clapham 1
PMCID: PMC281855  PMID: 2457901

Abstract

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.

Full text

PDF

Images in this article

5817Image
on p.5817

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnbaumer L., Brown A. M. G protein opening of K+ channels. Nature. 1987 May 7;327(6117):21–22. doi: 10.1038/327021a0. [DOI] [PubMed] [Google Scholar]
  2. Breitwieser G. E., Szabo G. Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature. 1985 Oct 10;317(6037):538–540. doi: 10.1038/317538a0. [DOI] [PubMed] [Google Scholar]
  3. Carmeliet E., Mubagwa K. Desensitization of the acetylcholine-induced increase of potassium conductance in rabbit cardiac Purkinje fibres. J Physiol. 1986 Feb;371:239–255. doi: 10.1113/jphysiol.1986.sp015971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Codina J., Yatani A., Grenet D., Brown A. M., Birnbaumer L. The alpha subunit of the GTP binding protein Gk opens atrial potassium channels. Science. 1987 Apr 24;236(4800):442–445. doi: 10.1126/science.2436299. [DOI] [PubMed] [Google Scholar]
  5. Evans T., Fawzi A., Fraser E. D., Brown M. L., Northup J. K. Purification of a beta 35 form of the beta gamma complex common to G-proteins from human placental membranes. J Biol Chem. 1987 Jan 5;262(1):176–181. [PubMed] [Google Scholar]
  6. Fung B. K., Hurley J. B., Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A. 1981 Jan;78(1):152–156. doi: 10.1073/pnas.78.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  8. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  9. Higashijima T., Ferguson K. M., Smigel M. D., Gilman A. G. The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. J Biol Chem. 1987 Jan 15;262(2):757–761. [PubMed] [Google Scholar]
  10. Huff R. M., Axton J. M., Neer E. J. Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex. J Biol Chem. 1985 Sep 5;260(19):10864–10871. [PubMed] [Google Scholar]
  11. Jelsema C. L., Axelrod J. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3623–3627. doi: 10.1073/pnas.84.11.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kurachi Y., Nakajima T., Sugimoto T. On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pflugers Arch. 1986 Sep;407(3):264–274. doi: 10.1007/BF00585301. [DOI] [PubMed] [Google Scholar]
  13. Kurachi Y., Nakajima T., Sugimoto T. Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins. Pflugers Arch. 1987 Oct;410(3):227–233. doi: 10.1007/BF00580270. [DOI] [PubMed] [Google Scholar]
  14. Logothetis D. E., Kurachi Y., Galper J., Neer E. J., Clapham D. E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 1987 Jan 22;325(6102):321–326. doi: 10.1038/325321a0. [DOI] [PubMed] [Google Scholar]
  15. Neer E. J., Lok J. M., Wolf L. G. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984 Nov 25;259(22):14222–14229. [PubMed] [Google Scholar]
  16. Neer E. J., Wolf L. G., Gill D. M. The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification. Biochem J. 1987 Jan 15;241(2):325–336. doi: 10.1042/bj2410325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Northup J. K., Sternweis P. C., Gilman A. G. The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution, activity, and properties of the 35,000-dalton (beta) subunit. J Biol Chem. 1983 Sep 25;258(18):11361–11368. [PubMed] [Google Scholar]
  18. Pfaffinger P. J., Martin J. M., Hunter D. D., Nathanson N. M., Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature. 1985 Oct 10;317(6037):536–538. doi: 10.1038/317536a0. [DOI] [PubMed] [Google Scholar]
  19. Ross E. M., Gilman A. G. Resolution of some components of adenylate cyclase necessary for catalytic activity. J Biol Chem. 1977 Oct 25;252(20):6966–6969. [PubMed] [Google Scholar]
  20. Stryer L., Bourne H. R. G proteins: a family of signal transducers. Annu Rev Cell Biol. 1986;2:391–419. doi: 10.1146/annurev.cb.02.110186.002135. [DOI] [PubMed] [Google Scholar]
  21. Winslow J. W., Van Amsterdam J. R., Neer E. J. Conformations of the alpha 39, alpha 41, and beta.gamma components of brain guanine nucleotide-binding proteins. Analysis by limited proteolysis. J Biol Chem. 1986 Jun 5;261(16):7571–7579. [PubMed] [Google Scholar]
  22. Yatani A., Codina J., Brown A. M., Birnbaumer L. Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk. Science. 1987 Jan 9;235(4785):207–211. doi: 10.1126/science.2432660. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES