Skip to main content
NCBI home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1992 May 1;99(5):665–682. doi: 10.1085/jgp.99.5.665

Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK

PMCID: PMC2216620  PMID: 1607851

Abstract

The effects of various intracellular anions on the G protein (GK)- mediated activation of the muscarinic K+ (KACh) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5 microM) in the pipette, 1 microM GTP caused different magnitudes of KACh channel activation in internal solutions containing different anions. The order of potency of anions to induce the KACh channel activity at 0.5 microM ACh and 1 microM GTP was Cl- greater than or equal to Br- greater than 1-. In the SO4(2-) or aspartic acid internal solution, no channel openings were induced by 1 microM GTP with 0.5 microM ACh. In both the Cl- and SO4(2-) internal solutions (with 0.5 microM ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (Kd) was 0.2 microM in the Cl- and 10 microM in the SO4(2-) solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-o-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO4(2-) solutions; i.e., the Hill coefficient was approximately 3-4 and the Kd was approximately 0.06-0.08 microM in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO4(2-) solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of GK, resulting in higher sensitivity of the KACh channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (greater than 1 microM) or ATP (greater than 1 mM) alone caused activation of the KACh channel, while neither occurred in the SO4(2-) solution. These observations suggest that the activation of the KACh channel by the basal turn-on reaction of GK or by phosphate transfer to GK by nucleoside diphosphate- kinase may depend at least partly on the intracellular concentration of Cl-.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Bahinski A., Nairn A. C., Greengard P., Gadsby D. C. Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature. 1989 Aug 31;340(6236):718–721. doi: 10.1038/340718a0. [DOI] [PubMed] [Google Scholar]
  2. Breitwieser G. E., Szabo G. Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues. J Gen Physiol. 1988 Apr;91(4):469–493. doi: 10.1085/jgp.91.4.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Brown A. M., Birnbaumer L. Ionic channels and their regulation by G protein subunits. Annu Rev Physiol. 1990;52:197–213. doi: 10.1146/annurev.ph.52.030190.001213. [DOI] [PubMed] [Google Scholar]
  5. Downs R. W., Jr, Spiegel A. M., Singer M., Reen S., Aurbach G. D. Fluoride stimulation of adenylate cyclase is dependent on the guanine nucleotide regulatory protein. J Biol Chem. 1980 Feb 10;255(3):949–954. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Harvey R. D., Hume J. R. Autonomic regulation of a chloride current in heart. Science. 1989 May 26;244(4907):983–985. doi: 10.1126/science.2543073. [DOI] [PubMed] [Google Scholar]
  9. Heidbüchel H., Callewaert G., Vereecke J., Carmeliet E. ATP-dependent activation of atrial muscarinic K+ channels in the absence of agonist and G-nucleotides. Pflugers Arch. 1990 Apr;416(1-2):213–215. doi: 10.1007/BF00370246. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Higashijima T., Ferguson K. M., Sternweis P. C. Regulation of hormone-sensitive GTP-dependent regulatory proteins by chloride. J Biol Chem. 1987 Mar 15;262(8):3597–3602. [PubMed] [Google Scholar]
  12. Ito H., Sugimoto T., Kobayashi I., Takahashi K., Katada T., Ui M., Kurachi Y. On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart. J Gen Physiol. 1991 Sep;98(3):517–533. doi: 10.1085/jgp.98.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ito H., Takikawa R., Iguchi M., Hamada E., Sugimoto T., Kurachi Y. Heparin uncouples the muscarinic receptors from GK protein in the atrial cell membrane of the guinea-pig heart. Pflugers Arch. 1990 Sep;417(1):126–128. doi: 10.1007/BF00370783. [DOI] [PubMed] [Google Scholar]
  14. Kaibara M., Nakajima T., Irisawa H., Giles W. Regulation of spontaneous opening of muscarinic K+ channels in rabbit atrium. J Physiol. 1991 Feb;433:589–613. doi: 10.1113/jphysiol.1991.sp018445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kikkawa S., Takahashi K., Takahashi K., Shimada N., Ui M., Kimura N., Katada T. Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins. J Biol Chem. 1990 Dec 15;265(35):21536–21540. [PubMed] [Google Scholar]
  16. Kimura N., Shimada N. Membrane-associated nucleoside diphosphate kinase from rat liver. Purification, characterization, and comparison with cytosolic enzyme. J Biol Chem. 1988 Apr 5;263(10):4647–4653. [PubMed] [Google Scholar]
  17. Kurachi Y., Ito H., Sugimoto T. Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP. Pflugers Arch. 1990 Apr;416(1-2):216–218. doi: 10.1007/BF00370247. [DOI] [PubMed] [Google Scholar]
  18. Kurachi Y., Nakajima T., Sugimoto T. Acetylcholine activation of K+ channels in cell-free membrane of atrial cells. Am J Physiol. 1986 Sep;251(3 Pt 2):H681–H684. doi: 10.1152/ajpheart.1986.251.3.H681. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kurachi Y., Nakajima T., Sugimoto T. Role of intracellular Mg2+ in the activation of muscarinic K+ channel in cardiac atrial cell membrane. Pflugers Arch. 1986 Nov;407(5):572–574. doi: 10.1007/BF00657521. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Neer E. J., Clapham D. E. Roles of G protein subunits in transmembrane signalling. Nature. 1988 May 12;333(6169):129–134. doi: 10.1038/333129a0. [DOI] [PubMed] [Google Scholar]
  24. Northup J. K., Smigel M. D., Sternweis P. C., Gilman A. G. The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit. J Biol Chem. 1983 Sep 25;258(18):11369–11376. [PubMed] [Google Scholar]
  25. Okabe K., Yatani A., Brown A. M. The nature and origin of spontaneous noise in G protein-gated ion channels. J Gen Physiol. 1991 Jun;97(6):1279–1293. doi: 10.1085/jgp.97.6.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Otero A. S., Breitwieser G. E., Szabo G. Activation of muscarinic potassium currents by ATP gamma S in atrial cells. Science. 1988 Oct 21;242(4877):443–445. doi: 10.1126/science.3051383. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Sternweis P. C. The purified alpha subunits of Go and Gi from bovine brain require beta gamma for association with phospholipid vesicles. J Biol Chem. 1986 Jan 15;261(2):631–637. [PubMed] [Google Scholar]
  29. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  30. Yamaoka K., Tanigawara Y., Nakagawa T., Uno T. A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn. 1981 Nov;4(11):879–885. doi: 10.1248/bpb1978.4.879. [DOI] [PubMed] [Google Scholar]
  31. Yatani A., Brown A. M. Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels. J Biol Chem. 1991 Dec 5;266(34):22872–22877. [PubMed] [Google Scholar]
  32. Yatani A., Okabe K., Polakis P., Halenbeck R., McCormick F., Brown A. M. ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell. 1990 Jun 1;61(5):769–776. doi: 10.1016/0092-8674(90)90187-j. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES