Advances in Bioscience and Biotechnology, 2013, 4, 665-673 ABB doi:10.4236/abb.2013.45087 Published Online May 2013 (http://www.scirp.org/journal/abb/ ) The action of ethanol on G protein. In silico and cellular/molecular evidences Pamela Fernandez 1 , Jessica Moreno 1,2 , Claudio Barrientos 1 , Sergio A. Aguila 3 , Daniela Leon 1,4 , Sebastián Ortiz 1 , Ramon Silva 1 , Francisco Rodriguez 1 , Maritza Leonardi 1 , Violeta Morin 2 , Ximena Romo 1* 1 Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Universidad Andres Bello Sede Concepcion, Talcahuano, Chile 2 Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepción, Concepcion, Chile 3 Universidad Nacional Autonoma de Mexico, Centro de Nanociencias y Nanotecnologia, Ensenada, Mexico 4 Laboratorio de Virus Oncológicos en Reproducción, BIOREN-CEGIN, Universidad de La Frontera, Temuco, Chile Email: * xromo@unab.cl Received 28 March 2013; revised 1 May 2013; accepted 21 May 2013 Copyright © 2013 Pamela Fernandez et al. This is an open access article distributed under the Creative Commons Attribution Li- cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 investi- gate the interaction between Gβγ and α1GlyR in re- sponse 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 etha- nol or propofol revealed that only EtOH treatment increased the binding of Gβγ heterodimers to α1GlyR. Using molecular modelling (protein structure predic- tion), 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 in- teraction between α1GlyR and Gβγ in HEK293 cells, an effect that might be attributed to the interaction between EtOH and hGαs, which consequently stimu- lates hGαs. Keywords: Alcoholism; Ethanol; Glycine Receptor; G Proteins; Signals Transduction 1. INTRODUCTION Ethanol (EtOH) is one of the most consumed drugs in the world [1]. EtOH acts specifically on many proteins [2] and as an inhibitory drug in the CNS by potentiating hy- perpolarising currents [3]. The α1 subunit of the Glycine Receptor (1GlyR) is an inhibitory receptor that is af- fected by EtOH. This is a selective channel for Cl − and is expressed within the spinal cord and within some regions of the brain [4]. Clinically relevant concentrations of EtOH (1 - 100 mM) are known to enhance glycinergic currents [5], increasing the rates of channel aperture. There are currently two proposed mechanisms of ac- tion of EtOH on α1GlyR. In the first mechanism, EtOH directly interacts with 1GlyR [6,7], and in the second mechanism, the action of EtOH on 1GlyR is exerted indirectly through its effects on Gβγ [8]. Consistent with the first proposed mechanism, several studies have re- ported that mutations on S687 and A288 of α1GlyR can eliminate its responsiveness to 200 mM EtOH, suggest- ing that the binding sites for EtOH are specific amino acids located within the TM2 and TM3 regions of 1GlyR. Some studies have reported that mutations can dramati- cally alter the functional properties of 1GlyR [9]. The discovery that 1GlyR directly interacts with Gβγ has provided secondary supporting evidence for these find- ings [10,11]. The α1GlyR variants that are mutated in ba- sic residues, which are considered key elements for the interaction between α1GlyR and Gβγ, were found to be insensitive to EtOH at clinically relevant concentrations (1 - 100 mM) [8]. In addition, the potentiation of glyci- nergic currents that are mediated by EtOH was reverted * Corresponding author. Published Online May 2013 in SciRes. http://www.scirp.org/journal/abb