Biochimica et Biophysica Acta, 979 (1989)147-152 Elsevier 147 The role of chloride ions on the transport of glycine in plasma membrane vesicles from glial cells F. Zafra and C. Gim6nez Departamento de Biologla Molecular, Centro de Biologla Molecular. Facultad de Ciencias. Universidad Aut~nonm de Madrit~ Madrid (Spain) (Received 12 September1988) Key words: Glycinetransport:Glial cell~Chlorideion: Plasmamembrane vesicle The high.affinity transport system for glycine in plasma membrane vesicles from C6 glioma cells is dependent on Na + and also on the presence of CI- in the incubation medium. This anion requirement is relatively specific for CI -, since other anions are also capable of stimulating the glycine transport in the following order of decreasing efficacy: Ci - > Br - > SCN - --- ! - > NO3 > F-. Chloride ions raise the V,~ for transport and, to a lesser extent, act on the K,r The data provided by direct measurements of the coupling of sodium and chloride to the transport of glycine by using a kinetic approach suggest a stoic~ometry for the translocation cycle catalyzed by the glycine transporter of two sodium ions and one chloride ion per glycine zwitterion. lntrodnction During the past years, studies have provided growing evidence suggesting that glial cells of the central nervous system play an important role in the modulation of neuronal excimbifity via the control of the levels of neuroactive substances in the extracellular mifieu of neurons [1-3]. The reuptake of neurotransmitter sub- stances into presynaptie nerve endings or glial cells provides one way of clearing the extracellular space of potentially neuroactive substances and so constitutes an efficient mechanism by which postsynaptic action can be terminated. Na+-dependent transport systems located in mam- malian plasma membranes are generally accepted to function by cotransport of substrate and Na +, and it has become clear that the electrochemical potential created by a Na + gradient serves as a direct driving force for the process [4-6]. A growing number of Na +- dependent transport systems also require Cl - for activ- ity, suggesting that CI- might also be cotransported [7-9]. Recently, we have reported the existence of two efficient uptake systems for glycine in plasma mem- brane vesicles derived from glioma cells; one is sodium- Abbreviations: SITS. 4-acetamido-4"-isothiocyanostilbene-2.2'-di- sulphonicacid; DMEM.Dulbecco's modified Eagle'smedium. Correspondence: C. Gimrnez. Centro de Biologia Molecular. Facul- tad de Ciencias. UniversidadAut6nomade Madrid. 28049 Madrid, Spain. 0005-2736/89/$03.50 © 1989 Elsevier Science PublishersB.V.(Biomedical and chloride-dependent with high affinity for the sub- strate, whereas the other has been shown to be Na+-de - pendent and Cl--independent with low affinity for glycine. The former shows kinetic features and energetic requirements similar to those found in nerve terminals [10,111. Considerable evidence now indicate that glycine, besides its role in numerous metabolic functions [12], acts as an inhibitory neurotransmitter in the mam- malian CNS, mainly in the spinal cord [13], and prob- ably in some localized areas of the brain, such as substantia nigra [14]. On the other hand, membrane vesicles isolated from various bacterial and mammalian cells, including neurons [15,16], and specifically from the glial plasma membrane [10], have been shown to be extremely useful in studying the amino-acid transport mechanisms, entailing a well-defined ion environment and energy sources and avoiding metabolic and com- partmentation interferences. The present paper extends previous data from our laboratory on glycine transport in glial cells in order to obtain a more detailed understanding about the mode of action of CI- in the Na+-coupled transporting sys- tems for amino-acid neurotransmitters. Materials and Methods Materials [U-14C]Glycine (113 mCi/mmol) was obtained from Amersham International, U.K., DMEM and fetal calf serum were obtained from Gibco, Paisley, U.K. Dishes Division)