The action of ethanol on G protein. In silico and cellular/molecular evidences

Pamela Fernandez, Jessica Moreno, Claudio Barrientos, Sergio A. Aguila, Daniela Leon, Sebastián Ortiz, Ramon Silva, Francisco Rodriguez, Maritza Leonardi, Violeta Morin, Ximena Romo

Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepción, Concepcion, Chile.
Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Universidad Andres Bello Sede Concepcion, Talcahuano, Chile.
Laboratorio de Virus Oncológicos en Reproducción, BIOREN-CEGIN, Universidad de La Frontera, Temuco, Chile.
Universidad Nacional Autonoma de Mexico, Centro de Nanociencias y Nanotecnologia, Ensenada, Mexico.

DOI: 10.4236/abb.2013.45087                

Abstract

Ethanol (EtOH) enhances glycinergic currents in the central nervous system (CNS). Because evidence for an interaction between the α1 subunit of the glycine receptor (α1GlyR) and the G protein Gβγ subunit exists in vitro and because cAMP levels are known to increase in response to EtOH, we wanted to investigate the interaction between Gβγ and α1GlyR in response to EtOH treatment in HEK293 cells and to explore the possible sites of interaction between EtOH and the Gαs subunit. His pull-down assays in GlyR-His6-transfected HEK293 cells incubated with ethanol or propofol revealed that only EtOH treatment increased the binding of Gβγ heterodimers to α1GlyR. Using molecular modelling (protein structure prediction), was modelled the hGαs protein for the first time and validated this model by site-directed mutagenesis. By molecular docking, we identified some potential regions of interaction between hGαs and EtOH that are located on the SIII and SI regions of the Gαs. Therefore, we conclude that ethanol increases the interaction between α1GlyR and Gβγ in HEK293 cells, an effect that might be attributed to the interaction between EtOH and hGαs, which consequently stimulates hGαs.

Keywords

Alcoholism; Ethanol; Glycine Receptor; G Proteins; Signals Transduction

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Hunt, W.A. (1993) Neuroscience research: How has it contributed to our understanding of alcohol abuse and alcoholism? A review, Alcohol. Clinical and Experimental Research, 17, 1055-1065. doi:10.1111/j.1530-0277.1993.tb05664.x
[2]	Harris, R.A., Trudell, J.R. and Mihic, S.J. (2008) Ethanol’s molecular targets. Science Signal, 1, re7. doi:10.1126/scisignal.128re7
[3]	Howard, R.J., Murail, S., Ondricek, K.E., Corringer, P.-J., Lindahl, E., Trudell, J.R., et al. (2011) Structural basis for alcohol modulation of a pentameric ligand-gated ion channel. Proceedings of National Academy Science of the USA, 108, 12149-12154. doi:10.1073/pnas.1104480108
[4]	Yevenes, G.E. and Zeilhofer, H.U. (2011) Allosteric modulation of glycine receptors. British Journal of Pharmacology, 164, 224-236. doi:10.1111/j.1476-5381.2011.01471.x
[5]	Aguayo, L.G. and Pancetti, F.C. (1994) Ethanol modulation of the gamma-aminobutyric acidAand glycine-activated Cl-current in cultured mouse neurons. Journal of Pharmacology and Experimental Therapeutics, 270, 6169.
[6]	Mihic, S.J., Ye, Q., Wick, M.J., Koltchine, V.V., Krasowski, M.D., Finn, S.E., et al. (1997) Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine recaptors. Nature, 389, 385-389. doi:10.1038/38738
[7]	Mascia, M.P., Trudell, J.R. and Harris, R.A. (2000) Specific binding sites for alcohols and anesthetics on ligandgated ion channels. Proceedings of National Academy Science of the USA, 97, 9305-9310. doi:10.1073/pnas.160128797
[8]	Yevenes, G.E., Moraga-Cid, G., Peoples, R.W., Schmalzing, G. and Aguayo, L.G. (2008) A selective Gβγ-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol. PNAS, 105, 20523-20528. doi:10.1073/pnas.0806257105
[9]	Findlay, G.S., Phelan, R., Roberts, M.T., Homanics, G.E., Bergeson, S.E., Lopreato, G.F., et al. (2003) Glycine receptor knock-in mice and hyperekplexia-like phenotypes: Comparisons with the null mutant. Journal of Neuroscience, 23, 8051-8059.
[10]	Yevenes, G.E., Peoples, R.W., Tapia, J.C., Parodi, J., Soto, X., Olate, J., et al. (2003) Modulation of glycine-activated ion channel function by G-protein betagamma subunits. Natural Neuroscience, 6, 819-824. doi:10.1038/nn1095
[11]	Yevenes, G.E., Moraga-Cid, G., Avila, A., Guzmán, L., Figueroa, M., Peoples, R.W., et al. (2010) Molecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gbetagamma modulation. Journal of Biological Chemistry, 285, 3020330213. doi:10.1074/jbc.M110.134676
[12]	Miyamoto, A., Yang, S.X., Laufs, U., Ruan, X.L. and Liao, J.K. (1999) Activation of guanine nucleotide-binding proteins and induction of endothelial tissue-type plasminogen activator gene transcription by alcohol. Journal of Biological Chemistry, 274, 12055-12060. doi:10.1074/jbc.274.17.12055
[13]	Constantinescu, A., Gordon, A.S. and Diamond, I. (2002) cAMP-dependent protein kinase types I and II differenttially regulate cAMP response element-mediated gene expression: Implications for neuronal responses to ethanol. Journal of Biological Chemistry, 277, 18810-18816. doi:10.1074/jbc.M112107200
[14]	Yoshimura, M. and Tabakoff, B. (1999) Ethanol’s actions on cAMP-mediated signaling in cells transfected with type VII adenylyl cyclase, Alcoholism: Clinical and Experimental Research, 23, 1457-1461.
[15]	Nelson, E.J., Hellevuo, K., Yoshimura, M. and Tabakoff, B. (2003) Ethanol-induced phosphorylation and potentialtion of the activity of type 7 adenylyl cyclase. Involvement of protein kinase C delta. Journal of Biological Chemistry, 278, 4552-4560. doi:10.1074/jbc.M210386200
[16]	Guzmán, L., Romo, X., Grandy, R., Soto, X., Montecino, M., Hinrichs, M., et al. (2005) A Gbetagamma stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation. Journal of Cellular Physiology, 202, 223-229. doi:10.1002/jcp.20102
[17]	Yevenes, G.E., Moraga-Cid, G., Romo, X. and Aguayo, L.G. (2011) Activated G protein (al-pha)s subunits increase the ethanol sensitivity of human glycine receptors. The Journal of Pharmacology and Experimental Therapeutics, 339, 386-393. doi:10.1124/jpet.111.184408
[18]	Lee, E., Linder, M.E. and Gilman, A.G. (1994) Expression of G-protein alpha subunits in Escherichia coli. Methods in Enzymology, 237, 146-164. doi:10.1016/S0076-6879(94)37059-1
[19]	Sprang, S.R. (1997) G protein mechanisms: Insights from structural analysis. Annual Review of Biochemistry, 66, 639-678. doi:10.1146/annurev.biochem.66.1.639
[20]	Brito, M., Guzmán, L., Romo, X., Soto, X., Hinrichs, M.V. and Olate, J. (2002) S111N mutation in the helical domain of human Gs(alpha) reduces its GDP/GTP exchange rate. Journal of Cellular Physiology, 85, 615-620. doi:10.1002/jcb.10128
[21]	Van Eps, N., Preininger, A.M., Alexander, N., Kaya, A.I., Meier, S., Meiler, J., et al. (2011) Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proceedings of National Academy Science of the USA, 108, 9420-9424. doi:10.1073/pnas.1105810108
[22]	Kapoor, N., Menon, S.T., Chauhan, R., Sachdev, P. and Sakmar, T.P. (2009) Structural evidence for a sequential release mechanism for activation of heterotrimeric G proteins. Journal of Molecular Biology, 393, 882-897. doi:10.1016/j.jmb.2009.08.043
[23]	Yevenes, G.E., Moraga-Cid, G., Peoples, R.W., Schmalzing, G. and Aguayo, L.G. (2008) A selective G betagamma-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol. Proceedings of National Academy Science of the USA, 105, 20523-20528. doi:10.1073/pnas.0806257105
[24]	Beck, I.T. and Dinda, P.K. (1981) Acute exposure of small intestine to ethanol: Effects on morphology and function. Digestive Diseases and Sciences, 26, 817-838. doi:10.1007/BF01309614
[25]	Dutta, S., Matsumoto, Y., Muramatsu, A., Matsumoto, M., Fukuoka, M. and Ebling, W.F. (1998) Steady-state propofol brain: Plasma and brain: Blood partition coefficients and the effect-site equilibration paradox. British Journal of Anaesthesia, 81, 422-424. doi:10.1093/bja/81.3.422
[26]	Tall, G.G. (2013) Ric-8 regulation of heterotrimeric G proteins. Journal of Receptors and Signal Transduction. doi:10.3109/10799893.2013.763828
[27]	Klattenhoff, C., Montecino, M., Soto, X., Guzmán, L., Romo, X., García, M.A., et al. (2003) Human brain synembryn interacts with Gsalpha and Gqalpha and is translocated to the plasma membrane in response to isoproterenol and carbachol. Journal of Cellular Physiology, 195, 151-157. doi:10.1002/jcp.10300
[28]	Romo, X., Pastén, P., Martínez, S., Soto, X., Lara, P., de Arellano, A.R., et al. (2008) xRic-8 is a GEF for Gsalpha and participates in maintaining meiotic arrest in Xenopus laevis oocytes. Journal of Cellular Physiology, 214, 673680. doi:10.1002/jcp.21257