Journal of Molecular Biology Research; Vol. 3, No. 1; 2013 ISSN 1925-430X E-ISSN 1925-4318 Published by Canadian Center of Science and Education 68 Expression of Human Chloride Channels ClC1 or ClC2 Revert the Petite Phenotype of a Saccharomyces cerevisiae GEF1 Mutant Fernando Rosas-Sanchez 1 , Angélica López-Rodríguez 1,2 , Carlos Saldaña 3 , Lenin Ochoa-de la Paz 1 , Ricardo Miledi 1 & Ataúlfo Martínez-Torres 1 1 Instituto de Neurobiología, Campus Juriquilla - Universidad Nacional Autónoma de México, Querétaro, México 2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA 3 Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, México Correspondence: Ataúlfo Martínez-Torres, Departamento de Neurobiología Molecular y Celular, Laboratorio de Neurobiología Molecular y Celular II, Instituto de Neurobiología, Campus Juriquilla-Universidad Nacional Autónoma de México. Boulevard Universitario 3001, C.P. 76230 Juriquilla, Querétaro, México. E-mail: ataulfo@unam.mx Received: December 28, 2012 Accepted: June 13, 2013 Online Published: July 2, 2013 doi:10.5539/jmbr.v3n1p68 URL: http://dx.doi.org/10.5539/jmbr.v3n1p68 Abstract The mechanism of activation of the yeast ClC chloride channel/transporter GEF1 is unknown, and in this study we tested the ability of human ClC1 and ClC2, two channels with different activation kinetics, to revert the petite phenotype of a strain whose GEF1 gene was deleted. We found that when the human channels are expressed in a low-copy plasmid, the reversion of the phenotype does not occur; in contrast, when the channels are over expressed by means of a strong transcriptional promoter in a multiple-copy plasmid, the cells reach the normal size, and show a normal membrane surface and oxygen consumption. To determine the size variationsof individual cells, we employed flow-cytometry as a quantitative tool to evaluate the petite phenotype. These results suggest that the human ClC channels, when abundantly present in the cells, can support the metabolism disrupted in the knock-out strain. We also observed that the fluorescence emitted by GFP-tagged channels was found mostly towards the periphery of the wt yeast, whereas in the GEF1 knock-out it was detected in intracellular clusters. GFP-tagged channels expressed in X. laevis oocytes produced robust currents and did not show any evident difference with respect to the normal ClCs, whereas Gep1p did not show voltage-dependent activation. Keywords: chloride channel, functional complementation, voltage-clamp, Xenopus laevis oocytes 1. Introduction Chloride channels/transporters (ClCs) are members of a large family present in a wide variety of organisms from bacteria to higher eukaryotes. ClCs carry out multiple physiological roles, from plasma membrane and cell volume modulation to the control of vesicular pH (Fahlke, 2001; Jentsch, Stein, Winreich & Zdebik, 2002; Sardini et al., 2003; Soleimani & Xu, 2006; Jentsch, 2008). A clear example of this functional diversification is illustrated by comparing the properties of mammalian ClC1 and ClC2. They are both located in the plasma membrane; however, whereas ClC1 is activated by plasma membrane depolarization and thus is responsible for the repolarization current in muscle fibers, ClC2 is activated by hyperpolarization, as well as by other mechanisms such as changes in pH and cell volume (Conte, De Luca, Mamrini, & Vrbovà, 1989; Steinmeyer, Ortland, & Jentsch, 1991; Klocke, Steinmeyer, Jentsch, & Jockusch, 1994; Jordt & Jentsch, 1997). The mechanism of activation of the Saccharomyces cerevisiae Gef1p, the sole ClC found in this species of yeast, is still not clearly understood. Gef1p plays a critical role in yeast iron metabolism and is found mainly in the trans-Golgi (Greene, Brown, DiDomenico, Kaplan & Eide, 1993; Schwappach, Stobrawa, Hechenberger, Steinmeyer & Jentsch, 1998). Mutations of the GEF1 gene lead to an iron requirement for growth on non-fermentable carbon sources due to a failure to load copper onto the iron uptake system; thus, knocking down the expression of GEF1 leads to petite (pet) colonies when grown in these conditions (Gaxiola et al., 1998). Gef1p forms a Cl - transporter/channel in the plasma membrane of the yeast that does not show