Abstract
Three mutations in the M2 transmembrane domains of the chloride-conducting alpha1 homomeric glycine receptor (P250Delta, A251E, and T265V), which normally mediate fast inhibitory neurotransmission, produced a cation-selective channel with P(Cl)/P(Na), = 0.27 (wild-type P(Cl)/P(Na) = 25), a permeability sequence P(Cs) > P(K) > P(Na) > P(Li), an impermeability to Ca(2+), and a reduced glycine sensitivity. Outside-out patch measurements indicated reversed and accentuated rectification with extremely low mean single channel conductances of 3 pS (inward current) and 11 pS (outward current). The three inverse mutations, to those analyzed in this study, have previously been shown to make the alpha7 acetylcholine receptor channel anion-selective, indicating a common location for determinants of charge selectivity of inhibitory and excitatory ligand-gated ion channels.
Full Text
The Full Text of this article is available as a PDF (175.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams D. J., Dwyer T. M., Hille B. The permeability of endplate channels to monovalent and divalent metal cations. J Gen Physiol. 1980 May;75(5):493–510. doi: 10.1085/jgp.75.5.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barry P. H. JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods. 1994 Jan;51(1):107–116. doi: 10.1016/0165-0270(94)90031-0. [DOI] [PubMed] [Google Scholar]
- Barry P. H., Lynch J. W. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol. 1991 Apr;121(2):101–117. doi: 10.1007/BF01870526. [DOI] [PubMed] [Google Scholar]
- Bertrand D., Galzi J. L., Devillers-Thiéry A., Bertrand S., Changeux J. P. Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6971–6975. doi: 10.1073/pnas.90.15.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bormann J., Hamill O. P., Sakmann B. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J Physiol. 1987 Apr;385:243–286. doi: 10.1113/jphysiol.1987.sp016493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bormann J., Rundström N., Betz H., Langosch D. Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers. EMBO J. 1993 Oct;12(10):3729–3737. doi: 10.1002/j.1460-2075.1993.tb06050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Corringer P. J., Bertrand S., Galzi J. L., Devillers-Thiéry A., Changeux J. P., Bertrand D. Mutational analysis of the charge selectivity filter of the alpha7 nicotinic acetylcholine receptor. Neuron. 1999 Apr;22(4):831–843. doi: 10.1016/s0896-6273(00)80741-2. [DOI] [PubMed] [Google Scholar]
- Dani J. A. Ion-channel entrances influence permeation. Net charge, size, shape, and binding considerations. Biophys J. 1986 Mar;49(3):607–618. doi: 10.1016/S0006-3495(86)83688-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eisenman G., Horn R. Ionic selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channels. J Membr Biol. 1983;76(3):197–225. doi: 10.1007/BF01870364. [DOI] [PubMed] [Google Scholar]
- Fatima-Shad K., Barry P. H. Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. Proc Biol Sci. 1993 Jul 22;253(1336):69–75. doi: 10.1098/rspb.1993.0083. [DOI] [PubMed] [Google Scholar]
- Galzi J. L., Devillers-Thiéry A., Hussy N., Bertrand S., Changeux J. P., Bertrand D. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature. 1992 Oct 8;359(6395):500–505. doi: 10.1038/359500a0. [DOI] [PubMed] [Google Scholar]
- Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
- Imoto K., Konno T., Nakai J., Wang F., Mishina M., Numa S. A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor. FEBS Lett. 1991 Sep 9;289(2):193–200. doi: 10.1016/0014-5793(91)81068-j. [DOI] [PubMed] [Google Scholar]
- Karlin A., Akabas M. H. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 1995 Dec;15(6):1231–1244. doi: 10.1016/0896-6273(95)90004-7. [DOI] [PubMed] [Google Scholar]
- Konno T., Busch C., Von Kitzing E., Imoto K., Wang F., Nakai J., Mishina M., Numa S., Sakmann B. Rings of anionic amino acids as structural determinants of ion selectivity in the acetylcholine receptor channel. Proc Biol Sci. 1991 May 22;244(1310):69–79. doi: 10.1098/rspb.1991.0053. [DOI] [PubMed] [Google Scholar]
- Kuhse J., Betz H., Kirsch J. The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex. Curr Opin Neurobiol. 1995 Jun;5(3):318–323. doi: 10.1016/0959-4388(95)80044-1. [DOI] [PubMed] [Google Scholar]
- Langosch D., Herbold A., Schmieden V., Borman J., Kirsch J. Importance of Arg-219 for correct biogenesis of alpha 1 homooligomeric glycine receptors. FEBS Lett. 1993 Dec 28;336(3):540–544. doi: 10.1016/0014-5793(93)80872-r. [DOI] [PubMed] [Google Scholar]
- Langosch D., Laube B., Rundström N., Schmieden V., Bormann J., Betz H. Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia. EMBO J. 1994 Sep 15;13(18):4223–4228. doi: 10.1002/j.1460-2075.1994.tb06742.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langosch D., Thomas L., Betz H. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7394–7398. doi: 10.1073/pnas.85.19.7394. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lynch J. W., Rajendra S., Pierce K. D., Handford C. A., Barry P. H., Schofield P. R. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO J. 1997 Jan 2;16(1):110–120. doi: 10.1093/emboj/16.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller C. Genetic manipulation of ion channels: a new approach to structure and mechanism. Neuron. 1989 Mar;2(3):1195–1205. doi: 10.1016/0896-6273(89)90304-8. [DOI] [PubMed] [Google Scholar]
- Moorhouse A. J., Jacques P., Barry P. H., Schofield P. R. The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating. Mol Pharmacol. 1999 Feb;55(2):386–395. doi: 10.1124/mol.55.2.386. [DOI] [PubMed] [Google Scholar]
- Pascual J. M., Karlin A. State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit. J Gen Physiol. 1998 Jun;111(6):717–739. doi: 10.1085/jgp.111.6.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quartararo N., Barry P. H., Gage P. W. Ion permeation through single channels activated by acetylcholine in denervated toad sartorius skeletal muscle fibers: effects of alkali cations. J Membr Biol. 1987;97(2):137–159. doi: 10.1007/BF01869420. [DOI] [PubMed] [Google Scholar]
- Rajendra S., Lynch J. W., Pierce K. D., French C. R., Barry P. H., Schofield P. R. Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists. Neuron. 1995 Jan;14(1):169–175. doi: 10.1016/0896-6273(95)90251-1. [DOI] [PubMed] [Google Scholar]
- Rajendra S., Lynch J. W., Pierce K. D., French C. R., Barry P. H., Schofield P. R. Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Biol Chem. 1994 Jul 22;269(29):18739–18742. [PubMed] [Google Scholar]
- Sansom M. S. Ion-channel gating. Twist to open. Curr Biol. 1995 Apr 1;5(4):373–375. doi: 10.1016/s0960-9822(95)00076-5. [DOI] [PubMed] [Google Scholar]
- Saul B., Kuner T., Sobetzko D., Brune W., Hanefeld F., Meinck H. M., Becker C. M. Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating. J Neurosci. 1999 Feb 1;19(3):869–877. doi: 10.1523/JNEUROSCI.19-03-00869.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tierney M. L., Birnir B., Cromer B., Howitt S. M., Gage P. W., Cox G. B. Two threonine residues in the M2 segment of the alpha 1 beta 1 GABAA receptor are critical for ion channel function. Receptors Channels. 1998;5(2):113–124. [PubMed] [Google Scholar]
- Unwin N. Acetylcholine receptor channel imaged in the open state. Nature. 1995 Jan 5;373(6509):37–43. doi: 10.1038/373037a0. [DOI] [PubMed] [Google Scholar]
- Villarroel A., Herlitze S., Witzemann V., Koenen M., Sakmann B. Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore. Proc Biol Sci. 1992 Sep 22;249(1326):317–324. doi: 10.1098/rspb.1992.0121. [DOI] [PubMed] [Google Scholar]
- Villarroel A., Sakmann B. Threonine in the selectivity filter of the acetylcholine receptor channel. Biophys J. 1992 Apr;62(1):196–208. doi: 10.1016/S0006-3495(92)81805-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang C. T., Zhang H. G., Rocheleau T. A., ffrench-Constant R. H., Jackson M. B. Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila. Biophys J. 1999 Aug;77(2):691–700. doi: 10.1016/S0006-3495(99)76924-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang F., Imoto K. Pore size and negative charge as structural determinants of permeability in the Torpedo nicotinic acetylcholine receptor channel. Proc Biol Sci. 1992 Oct 22;250(1327):11–17. doi: 10.1098/rspb.1992.0124. [DOI] [PubMed] [Google Scholar]
- Wilson G. G., Karlin A. The location of the gate in the acetylcholine receptor channel. Neuron. 1998 Jun;20(6):1269–1281. doi: 10.1016/s0896-6273(00)80506-1. [DOI] [PubMed] [Google Scholar]
- Wilson G. G., Pascual J. M., Brooijmans N., Murray D., Karlin A. The intrinsic electrostatic potential and the intermediate ring of charge in the acetylcholine receptor channel. J Gen Physiol. 2000 Feb;115(2):93–106. doi: 10.1085/jgp.115.2.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu M., Akabas M. H. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit. J Gen Physiol. 1996 Feb;107(2):195–205. doi: 10.1085/jgp.107.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu M., Covey D. F., Akabas M. H. Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants. Biophys J. 1995 Nov;69(5):1858–1867. doi: 10.1016/S0006-3495(95)80056-1. [DOI] [PMC free article] [PubMed] [Google Scholar]