Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Feb;80(2):707–718. doi: 10.1016/S0006-3495(01)76050-X

Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels.

P A Slesinger 1
PMCID: PMC1301269  PMID: 11159438

Abstract

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.

Full Text

The Full Text of this article is available as a PDF (171.3 KB).

Selected References

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

  1. Adams C. M., Price M. P., Snyder P. M., Welsh M. J. Tetraethylammonium block of the BNC1 channel. Biophys J. 1999 Mar;76(3):1377–1383. doi: 10.1016/S0006-3495(99)77299-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alreja M., Aghajanian G. K. QX-314 blocks the potassium but not the sodium-dependent component of the opiate response in locus coeruleus neurons. Brain Res. 1994 Mar 14;639(2):320–324. doi: 10.1016/0006-8993(94)91746-9. [DOI] [PubMed] [Google Scholar]
  3. Andrade R. Blockade of neurotransmitter-activated K+ conductance by QX-314 in the rat hippocampus. Eur J Pharmacol. 1991 Jun 25;199(2):259–262. doi: 10.1016/0014-2999(91)90467-5. [DOI] [PubMed] [Google Scholar]
  4. Baukrowitz T., Yellen G. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. Science. 1996 Feb 2;271(5249):653–656. doi: 10.1126/science.271.5249.653. [DOI] [PubMed] [Google Scholar]
  5. Chan K. W., Sui J. L., Vivaudou M., Logothetis D. E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14193–14198. doi: 10.1073/pnas.93.24.14193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choe S., Stevens C. F., Sullivan J. M. Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12046–12049. doi: 10.1073/pnas.92.26.12046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins A., Chuang H., Jan Y. N., Jan L. Y. Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5456–5460. doi: 10.1073/pnas.94.10.5456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doupnik C. A., Davidson N., Lester H. A. The inward rectifier potassium channel family. Curr Opin Neurobiol. 1995 Jun;5(3):268–277. doi: 10.1016/0959-4388(95)80038-7. [DOI] [PubMed] [Google Scholar]
  9. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  10. Duprat F., Lesage F., Guillemare E., Fink M., Hugnot J. P., Bigay J., Lazdunski M., Romey G., Barhanin J. Heterologous multimeric assembly is essential for K+ channel activity of neuronal and cardiac G-protein-activated inward rectifiers. Biochem Biophys Res Commun. 1995 Jul 17;212(2):657–663. doi: 10.1006/bbrc.1995.2019. [DOI] [PubMed] [Google Scholar]
  11. Hedin K. E., Lim N. F., Clapham D. E. Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes. Neuron. 1996 Feb;16(2):423–429. doi: 10.1016/s0896-6273(00)80060-4. [DOI] [PubMed] [Google Scholar]
  12. Heginbotham L., Lu Z., Abramson T., MacKinnon R. Mutations in the K+ channel signature sequence. Biophys J. 1994 Apr;66(4):1061–1067. doi: 10.1016/S0006-3495(94)80887-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang C. L., Slesinger P. A., Casey P. J., Jan Y. N., Jan L. Y. Evidence that direct binding of G beta gamma to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation. Neuron. 1995 Nov;15(5):1133–1143. doi: 10.1016/0896-6273(95)90101-9. [DOI] [PubMed] [Google Scholar]
  14. Hurst R. S., Latorre R., Toro L., Stefani E. External barium block of Shaker potassium channels: evidence for two binding sites. J Gen Physiol. 1995 Dec;106(6):1069–1087. doi: 10.1085/jgp.106.6.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Inanobe A., Morishige K. I., Takahashi N., Ito H., Yamada M., Takumi T., Nishina H., Takahashi K., Kanaho Y., Katada T. G beta gamma directly binds to the carboxyl terminus of the G protein-gated muscarinic K+ channel, GIRK1. Biochem Biophys Res Commun. 1995 Jul 26;212(3):1022–1028. doi: 10.1006/bbrc.1995.2072. [DOI] [PubMed] [Google Scholar]
  16. Kennedy M. E., Nemec J., Clapham D. E. Localization and interaction of epitope-tagged GIRK1 and CIR inward rectifier K+ channel subunits. Neuropharmacology. 1996;35(7):831–839. doi: 10.1016/0028-3908(96)00132-3. [DOI] [PubMed] [Google Scholar]
  17. Kim C. J., Kim H. O., Choe Y. J., Lee Y. A., Kim C. W. Bcl-2 expression in neuroblastoma is differentially regulated by differentiation inducers. Anticancer Res. 1995 Sep-Oct;15(5B):1997–2000. [PubMed] [Google Scholar]
  18. Kofuji P., Davidson N., Lester H. A. Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6542–6546. doi: 10.1073/pnas.92.14.6542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kofuji P., Doupnik C. A., Davidson N., Lester H. A. A unique P-region residue is required for slow voltage-dependent gating of a G protein-activated inward rectifier K+ channel expressed in Xenopus oocytes. J Physiol. 1996 Feb 1;490(Pt 3):633–645. doi: 10.1113/jphysiol.1996.sp021173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kofuji P., Hofer M., Millen K. J., Millonig J. H., Davidson N., Lester H. A., Hatten M. E. Functional analysis of the weaver mutant GIRK2 K+ channel and rescue of weaver granule cells. Neuron. 1996 May;16(5):941–952. doi: 10.1016/s0896-6273(00)80117-8. [DOI] [PubMed] [Google Scholar]
  21. Krapivinsky G., Gordon E. A., Wickman K., Velimirović B., Krapivinsky L., Clapham D. E. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins. Nature. 1995 Mar 9;374(6518):135–141. doi: 10.1038/374135a0. [DOI] [PubMed] [Google Scholar]
  22. Krapivinsky G., Krapivinsky L., Wickman K., Clapham D. E. G beta gamma binds directly to the G protein-gated K+ channel, IKACh. J Biol Chem. 1995 Dec 8;270(49):29059–29062. doi: 10.1074/jbc.270.49.29059. [DOI] [PubMed] [Google Scholar]
  23. Kraus R. L., Hering S., Grabner M., Ostler D., Striessnig J. Molecular mechanism of diltiazem interaction with L-type Ca2+ channels. J Biol Chem. 1998 Oct 16;273(42):27205–27212. doi: 10.1074/jbc.273.42.27205. [DOI] [PubMed] [Google Scholar]
  24. Kubo Y., Reuveny E., Slesinger P. A., Jan Y. N., Jan L. Y. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature. 1993 Aug 26;364(6440):802–806. doi: 10.1038/364802a0. [DOI] [PubMed] [Google Scholar]
  25. Kunkel M. T., Peralta E. G. Identification of domains conferring G protein regulation on inward rectifier potassium channels. Cell. 1995 Nov 3;83(3):443–449. doi: 10.1016/0092-8674(95)90122-1. [DOI] [PubMed] [Google Scholar]
  26. Lambert N. A., Wilson W. A. Discrimination of post- and presynaptic GABAB receptor-mediated responses by tetrahydroaminoacridine in area CA3 of the rat hippocampus. J Neurophysiol. 1993 Feb;69(2):630–635. doi: 10.1152/jn.1993.69.2.630. [DOI] [PubMed] [Google Scholar]
  27. Lesage F., Guillemare E., Fink M., Duprat F., Heurteaux C., Fosset M., Romey G., Barhanin J., Lazdunski M. Molecular properties of neuronal G-protein-activated inwardly rectifying K+ channels. J Biol Chem. 1995 Dec 1;270(48):28660–28667. doi: 10.1074/jbc.270.48.28660. [DOI] [PubMed] [Google Scholar]
  28. Loussouarn G., Makhina E. N., Rose T., Nichols C. G. Structure and dynamics of the pore of inwardly rectifying K(ATP) channels. J Biol Chem. 2000 Jan 14;275(2):1137–1144. doi: 10.1074/jbc.275.2.1137. [DOI] [PubMed] [Google Scholar]
  29. Lu T., Nguyen B., Zhang X., Yang J. Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification. Neuron. 1999 Mar;22(3):571–580. doi: 10.1016/s0896-6273(00)80711-4. [DOI] [PubMed] [Google Scholar]
  30. Lu Z., MacKinnon R. Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature. 1994 Sep 15;371(6494):243–246. doi: 10.1038/371243a0. [DOI] [PubMed] [Google Scholar]
  31. Minor D. L., Jr, Masseling S. J., Jan Y. N., Jan L. Y. Transmembrane structure of an inwardly rectifying potassium channel. Cell. 1999 Mar 19;96(6):879–891. doi: 10.1016/s0092-8674(00)80597-8. [DOI] [PubMed] [Google Scholar]
  32. Nathan T., Jensen M. S., Lambert J. D. The slow inhibitory postsynaptic potential in rat hippocampal CA1 neurones is blocked by intracellular injection of QX-314. Neurosci Lett. 1990 Mar 14;110(3):309–313. doi: 10.1016/0304-3940(90)90865-7. [DOI] [PubMed] [Google Scholar]
  33. Nicoll R. A., Malenka R. C., Kauer J. A. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol Rev. 1990 Apr;70(2):513–565. doi: 10.1152/physrev.1990.70.2.513. [DOI] [PubMed] [Google Scholar]
  34. North R. A. Twelfth Gaddum memorial lecture. Drug receptors and the inhibition of nerve cells. Br J Pharmacol. 1989 Sep;98(1):13–28. doi: 10.1111/j.1476-5381.1989.tb16855.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Otis T. S., De Koninck Y., Mody I. Characterization of synaptically elicited GABAB responses using patch-clamp recordings in rat hippocampal slices. J Physiol. 1993 Apr;463:391–407. doi: 10.1113/jphysiol.1993.sp019600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Patil N., Cox D. R., Bhat D., Faham M., Myers R. M., Peterson A. S. A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nat Genet. 1995 Oct;11(2):126–129. doi: 10.1038/ng1095-126. [DOI] [PubMed] [Google Scholar]
  37. Philipson L. H., Kuznetsov A., Toth P. T., Murphy J. F., Szabo G., Ma G. H., Miller R. J. Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1). J Biol Chem. 1995 Jun 16;270(24):14604–14610. doi: 10.1074/jbc.270.24.14604. [DOI] [PubMed] [Google Scholar]
  38. Ragsdale D. S., McPhee J. C., Scheuer T., Catterall W. A. Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9270–9275. doi: 10.1073/pnas.93.17.9270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reuveny E., Jan Y. N., Jan L. Y. Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation. Biophys J. 1996 Feb;70(2):754–761. doi: 10.1016/S0006-3495(96)79615-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reuveny E., Slesinger P. A., Inglese J., Morales J. M., Iñiguez-Lluhi J. A., Lefkowitz R. J., Bourne H. R., Jan Y. N., Jan L. Y. Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. Nature. 1994 Jul 14;370(6485):143–146. doi: 10.1038/370143a0. [DOI] [PubMed] [Google Scholar]
  41. Signorini S., Liao Y. J., Duncan S. A., Jan L. Y., Stoffel M. Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):923–927. doi: 10.1073/pnas.94.3.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Silverman S. K., Kofuji P., Dougherty D. A., Davidson N., Lester H. A. A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15429–15434. doi: 10.1073/pnas.93.26.15429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Silverman S. K., Lester H. A., Dougherty D. A. Asymmetrical contributions of subunit pore regions to ion selectivity in an inward rectifier K+ channel. Biophys J. 1998 Sep;75(3):1330–1339. doi: 10.1016/S0006-3495(98)74051-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Slesinger P. A., Patil N., Liao Y. J., Jan Y. N., Jan L. Y., Cox D. R. Functional effects of the mouse weaver mutation on G protein-gated inwardly rectifying K+ channels. Neuron. 1996 Feb;16(2):321–331. doi: 10.1016/s0896-6273(00)80050-1. [DOI] [PubMed] [Google Scholar]
  45. Slesinger P. A., Reuveny E., Jan Y. N., Jan L. Y. Identification of structural elements involved in G protein gating of the GIRK1 potassium channel. Neuron. 1995 Nov;15(5):1145–1156. doi: 10.1016/0896-6273(95)90102-7. [DOI] [PubMed] [Google Scholar]
  46. Slesinger P. A., Stoffel M., Jan Y. N., Jan L. Y. Defective gamma-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):12210–12217. doi: 10.1073/pnas.94.22.12210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sunami A., Dudley S. C., Jr, Fozzard H. A. Sodium channel selectivity filter regulates antiarrhythmic drug binding. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):14126–14131. doi: 10.1073/pnas.94.25.14126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. TRAUTWEIN W., DUDEL J. Zum Mechanismus der Membranwirkung des Acetylcholin an der Herzmuskelfaser. Pflugers Arch. 1958;266(3):324–334. doi: 10.1007/BF00416781. [DOI] [PubMed] [Google Scholar]
  49. Tucker S. J., Pessia M., Moorhouse A. J., Gribble F., Ashcroft F. M., Maylie J., Adelman J. P. Heteromeric channel formation and Ca(2+)-free media reduce the toxic effect of the weaver Kir 3.2 allele. FEBS Lett. 1996 Jul 29;390(3):253–257. doi: 10.1016/0014-5793(96)00635-7. [DOI] [PubMed] [Google Scholar]
  50. Velimirovic B. M., Gordon E. A., Lim N. F., Navarro B., Clapham D. E. The K+ channel inward rectifier subunits form a channel similar to neuronal G protein-gated K+ channel. FEBS Lett. 1996 Jan 22;379(1):31–37. doi: 10.1016/0014-5793(95)01465-9. [DOI] [PubMed] [Google Scholar]
  51. Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yamada K., Yu B., Gallagher J. P. Different subtypes of GABAB receptors are present at pre- and postsynaptic sites within the rat dorsolateral septal nucleus. J Neurophysiol. 1999 Jun;81(6):2875–2883. doi: 10.1152/jn.1999.81.6.2875. [DOI] [PubMed] [Google Scholar]
  53. Yellen G., Jurman M. E., Abramson T., MacKinnon R. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. Science. 1991 Feb 22;251(4996):939–942. doi: 10.1126/science.2000494. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES