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Expression and Functional Characterization of GABA Transporters in Crayfish Neurosecretory Cells

The effect of GABA on membrane potential and ionic currents of X-organ neurons isolated from the crayfish eyestalk was investigated. Under voltage-clamp conditions, GABA elicited an inward Na+ current followed by a sustained outward chloride current. Sodium current was partially blocked in a dose-dependent manner by antagonists of GABA plasma membrane transporters such as β-alanine, nipecotic acid, 1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711), and SKF89976-A at concentrations between 1 and 100 μm. This current was totally blocked by the combined application of NO 711 (5 μm) and β-alanine (50 μm). We obtained an EC50 of 5 μm and a Hill coefficient of 0.97 for the GABA transport mediated response. These results together with studies of immunolocalization using antibodies against neuronal vertebrate GABA transporters (GATs) indicate the presence of GAT-1- and GAT-3-like proteins in X-organ neurons. To isolate the sustained outward Cl− current, extracellular free sodium solution was used to minimize the contribution of GAT activity. We concluded that this current was caused by the activation of GABAA-like receptors with an EC50 of 10 μm and a Hill number of 1.7. To assign a functional role to the GATs in the X-organ sinus gland system, we determine the GABA concentration (0.46–0.15 μm) in hemolymph samples using HPLC. In summary, our results suggest that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the excitability of the X-organ neurons, maintaining an excitatory tone that is dependent on the circulating GABA level.