Characterization of the gamma-aminobutyric acid transporter present in the horizontal cells of the skate retina

An investigation of the ionic and pharmacological properties of the GABA transporter present in retinal interneurons was performed using an electrophysiological approach. The characterization of the membrane protein was accomplished through three experimental aspects. (1) Ionic Dependence and Stoichiometry; (2) Pharmacology; (3) Modulation.; Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter present in the vertebrate CNS. The termination of GABA synaptic transmission occurs by re-uptake into both neurons and glia via membrane proteins called transporters. In the retina of many vertebrate species, GABA is the inhibitory transmitter of some classes of horizontal cells.; The horizontal cells used in this investigation are those of a marine elasmobranch known as the skate. Horizontal cells are readily isolated from the all-rod skate retina, and are easily identified by their size and their morphology. Skate horizontal cells lack GABA receptors. Therefore, the large inward current produced from extracellular application of GABA onto isolated horizontal cells was solely due to the response of the GABA transporters. Using the whole-cell version of the patch-clamp recording technique, it was determined that the GABA transporter current in these cells was saturable at about 1 mM GABA, and did not desensitize or diminish with time. Two sodium ions and one chloride ion were required for GABA transport to occur.; Pharmacologically, skate horizontal cell GABA transporters were similar to the neuronal GABA transporter-1 (GAT-1) in their response to agonists and antagonist of GABA transport. Modulation of the GABA transporter current occurred by extracellular application of ATP and P2x purinoceptors agonists, with adenosine having no apparent effect. Modulation was dependent on the presence of calcium in the extracellular fluid, but could also be induced upon release of calcium from intracellular stores by caffeine and thapsigargin. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) also caused modulation of the GABA transporter current.