[en] Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the alpha1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gbetagamma with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC(50) (40 +/- 1 versus 41 +/- 0.5 muM) or the ethanol-induced potentiation (53 +/- 5 versus 46 +/- 5%) of the human alpha1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 +/- 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 +/- 4 and 17 +/- 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gbetagamma, is critical for ethanol potentiation of the receptor.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Castro, Patricio A
Figueroa, Maximiliano ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Yevenes, Gonzalo E
San Martin, Loreto S
Aguayo, Luis G
Language :
English
Title :
The Basic Property of Lys385 Is Important for Potentiation of the Human alpha1 Glycine Receptor by Ethanol.
Publication date :
2012
Journal title :
Journal of Pharmacology and Experimental Therapeutics
ISSN :
0022-3565
eISSN :
1521-0103
Publisher :
American Society for Pharmacology and Experimental Therapeutics, Bethesda, United States - Maryland
Aguayo LG and Pancetti FC (1994) Ethanol modulation of the γ-aminobutyric acid A- and glycine-activated Cl- current in cultured mouse neurons. J Pharmacol Exp Ther 270:61-69. (Pubitemid 2110403)
Aguayo LG, Tapia JC, and Pancetti FC (1996) Potentiation of the glycine-activated Cl - current by ethanol in cultured mouse spinal neurons. J Pharmacol Exp Ther 279:1116-1122.
Ahmadi S, Lippross S, Neuhuber WL, and Zeilhofer HU (2002) PGE 2 selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons. Nat Neurosci 5:34-40.
Anderson RJ, Weng Z, Campbell RK, and Jiang X (2005) Main-chain conformational tendencies of amino acids. Proteins 60:679-689. (Pubitemid 41266425)
Baker NA, Sept D, Joseph S, Holst MJ, and McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98:10037-10041. (Pubitemid 32802969)
Barr AJ, Ali H, Haribabu B, Snyderman R, and Smrcka AV (2000) Identification of a region at the N terminus of phospholipase C-β3 that interacts with G protein βγ subunits. Biochemistry 39:1800-1806. (Pubitemid 30108972)
Cantí C, Page KM, Stephens GJ, and Dolphin AC (1999) Identification of residues in the N terminus of α1B critical for inhibition of the voltage-dependent calcium channel by Gβγ. J Neurosci 19:6855-6864. (Pubitemid 29380114)
Carland JE, Cooper MA, Sugiharto S, Jeong HJ, Lewis TM, Barry PH, Peters JA, Lambert JJ, and Moorhouse AJ (2009) Characterization of the effects of charged residues in the intracellular loop on ion permeation in α1 glycine receptor channels. J Biol Chem 284:2023-2030.
Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB, and Sixma TK (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41:907-914. (Pubitemid 38429734)
Crawford DK, Trudell JR, Bertaccini EJ, Li K, Davies DL, and Alkana RL (2007) Evidence that ethanol acts on a target in loop 2 of the extracellular domain of α1 glycine receptors. J Neurochem 102:2097-2109. (Pubitemid 47329273)
Deeb TZ, Carland JE, Cooper MA, Livesey MR, Lambert JJ, Peters JA, and Hales TG (2007) Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor. J Biol Chem 282:6172-6182. (Pubitemid 47100876)
Dolinsky TJ, Czodrowski P, Li H, Nielsen JE, Jensen JH, Klebe G, and Baker NA (2007) PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res 35:W522-W525.
Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, and Sali A (2006) Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics, Chapter 5, Unit 5.6.
Fischer H, Liu DM, Lee A, Harries JC, and Adams DJ (2005) Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by Go-protein subunits. J Neurosci 25:3571-3577.
Ford CE, Skiba NP, Bae H, Daaka Y, Reuveny E, Shekter LR, Rosal R, Weng G, Yang CS, Iyengar R, et al. (1998) Molecular basis for interactions of G protein βγsubunits with effectors. Science 280:1271-1274. (Pubitemid 28269623)
Ginalski K, Elofsson A, Fischer D, and Rychlewski L (2003) 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19:1015-1018. (Pubitemid 36675823)
Godden EL, Harris RA, and Dunwiddie TV (2001) Correlation between molecular volume and effects of n-alcohols on human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 296:716-722. (Pubitemid 32187339)
Harris RA, Trudell JR, and Mihic SJ (2008) Ethanol's molecular targets. Sci Signal 1:re7.
Hassinen T and Peräkylä M (2001) New energy terms for reduced protein models implemented in an off-lattice force field. J Comput Chem 22:1229-1242. (Pubitemid 32763389)
Hess B, Kutzner C, van der Spoel D, and Lindahl E (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4:435-447.
Leckband D (2000) Measuring the forces that control protein interactions. Annu Rev Biophys Biomol Struct 29:1-26.
Legendre P (2001) The glycinergic inhibitory synapse. Cell Mol Life Sci 58:760-793.
Lester HA, Dibas MI, Dahan DS, Leite JF, and Dougherty DA (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27:329-336. (Pubitemid 38692414)
Li Y and Zhang Y (2009) REMO: a new protocol to refine full atomic protein models from C-α traces by optimizing hydrogen-bonding networks. Proteins 76:665-676.
Livesey MR, Cooper MA, Deeb TZ, Carland JE, Kozuska J, Hales TG, Lambert JJ, and Peters JA (2008) Structural determinants of Ca 2+ permeability and conduction in the human 5-hydroxytryptamine type 3A receptor. J Biol Chem 283:19301-19313.
Lovinger DM and White G (1991) Ethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in neuroblastoma and isolated adult mammalian neurons. Mol Pharmacol 40:263-270.
Lynch JW (2004) Molecular structure and function of the glycine receptor chloride channel. Physiol Rev 84:1051-1095.
Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, et al. (1997) Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature 389:385-389. (Pubitemid 27415220)
Perkins DI, Trudell JR, Crawford DK, Alkana RL, and Davies DL (2008) Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors. J Neurochem 106:1337-1349.
Peters JA, Cooper MA, Carland JE, Livesey MR, Hales TG, and Lambert JJ (2010) Novel structural determinants of single channel conductance and ion selectivity in 5-hydroxytryptamine type 3 and nicotinic acetylcholine receptors. J Physiol 588:587-596.
Popova M, Asatryan L, Ostrovskaya O, Wyatt LR, Li K, Alkana RL, and Davies DL (2010) A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. J Neurochem 112:307-317.
Ren H, Salous AK, Paul JM, Lamb KA, Dwyer DS, and Peoples RW (2008) Functional interactions of alcohol-sensitive sites in the N-methyl-D-aspartate receptor M3 and M4 domains. J Biol Chem 283:8250-8257.
Sadtler S, Laube B, Lashub A, Nicke A, Betz H, and Schmalzing G (2003) A basic cluster determines topology of the cytoplasmic M3-M4 loop of the glycine receptor α1 subunit. J Biol Chem 278:16782-16790. (Pubitemid 36799546)
Touhara K, Koch WJ, Hawes BE, and Lefkowitz RJ (1995) Mutational analysis of the pleckstrin homology domain of the β-adrenergic receptor kinase. Differential effects on Gβγ and phosphatidylinositol 4,5-bisphosphate binding. J Biol Chem 270:17000-17005.
Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4Åresolution. J Mol Biol 346:967-989.
Yamakura T, Mihic SJ, and Harris RA (1999) Amino acid volume and hydropathy of a transmembrane site determine glycine and anesthetic sensitivity of glycine receptors. J Biol Chem 274:23006-23012.
Ye Q, Koltchine VV, Mihic SJ, Mascia MP, Wick MJ, Finn SE, Harrison NL, and Harris RA (1998) Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position α267. J Biol Chem 273:3314-3319. (Pubitemid 28109745)
Yevenes GE, Moraga-Cid G, Avila A, Guzmán L, Figueroa M, Peoples RW, and Aguayo LG (2010) Molecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gβγ modulation. J Biol Chem285:30203-30213.
Yevenes GE, Moraga-Cid G, Guzmán L, Haeger S, Oliveira L, Olate J, Schmalzing G, and Aguayo LG (2006) Molecular determinants for G protein βγ modulation of ionotropic glycine receptors. J Biol Chem 281:39300-39307. (Pubitemid 46041889)
Yevenes GE, Moraga-Cid G, Peoples RW, Schmalzing G, and Aguayo LG (2008) A selective Gβγ-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol. Proc Natl Acad Sci U S A 105:20523-20528.
