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The Journal of Neuroscience, November 1, 2002, 22(21):9176-9184

Expression and Functional Characterization of GABA Transporters in Crayfish Neurosecretory Cells

Julieta Garduño, Sergio Elenes, Jorge Cebada, Elizabeth Becerra, and Ubaldo García

Department of Physiology, Biophysics, and Neuroscience, Centro de Investigación y de Estudios Avanzados, 07360 Mexico City, Mexico

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 -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 -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 GABA_A-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.

Key words: Procambarus clarkii; crustaceans; crayfish; X-organ sinus gland system; peptidergic neurons; GABA transporters; GABA_A receptors

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Copyright © 2002 Society for Neuroscience  0270-6474/02/22219176-09$05.00/0

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