Expression and Functional Characterization of GABA Transporters in Crayfish Neurosecretory Cells

Abstract

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 ϩ 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 M. This current was totally blocked by the combined application of NO 711 (5 M) and ␤-alanine (50 M). We obtained an EC 50 of 5 M 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 Ϫ 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 EC 50 of 10 M 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 M) 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.

Histamine operates Cl–-gated channels in crayfish neurosecretory cells

“…In the other 50% of the cells, Glu elicited a fully desensitizing current; similar results have been described previously (Duan and Cooke, 2000). By contrast, as previously reported in crayfish X-organ neurons (Garduño et al, 2002), GABA-evoked currents exhibited two components: an early transient inward current due to the electrogenic GABA uptake, followed by a sustained outward current generated through ligand-gated Cl -channels (Fig.·3A, middle trace). Finally, the HA-evoked current declined slowly during the continued application (Fig.·3A, right trace).…”

“…The HA-induced current was measured at the peak and these values were plotted against the log of the HA concentration, giving a concentration inducing a half-maximal response (EC 50 ) of 3.28±1·mol·l -1 and Hill number of 2.6±0.4 (Fig.·4B). The EC 50 value for HA differs considerably from those reported for Glu and GABA (Duan and Cooke, 2000;Garduño et al, 2002). Previous work has shown that the homomeric expression of the mRNAs encoding HA-gated Cl -channel subunits from the fruit fly Drosophila melanogaster produced HA receptors with EC 50 values of 166±12·mol·l -1 for DMHisCl-␣1 and 10.8±0.46 DM-HisCl-␣2 (Gisselmann et al, 2002), whereas those for HisCl-1 and HisCl-2 were 4.2±1.3 and 14±2.5, respectively (Zheng et al, 2002).…”

“…The presence of immunoreactive neurons to histamine in the medulla terminalis with axonal projections to the neuropile suggests a possible histaminergic modulation of the X-organ sinus gland system. activation of a Na + -dependent inward current associated with the GABA uptake, that is followed by an inhibitory phase due to the activation of a Cl -dependent outward current which is associated with the activation of GABA A -like receptors (Garduño et al, 2002).…”

Subcellular redistribution of the renal betaine transporter during hypertonic stress

“…Nipecotic acid, a cyclic analog of GABA, and ␤-alanine are competitive inhibitors of GABA transporters (14) and likely bind to the same site as the GABA (or betaine) substrate (46). These results suggest that the conformational stability of the transport site, or the fidelity of the transport function, is not required for the signals that regulate insertion of protein into the plasma membrane.…”

“…C3H/10T 1 ⁄2 mouse embryo fibroblasts (43) (CCL-226; American Type Culture Collection) were cultured in DMEM containing 10% heat-inactivated fetal bovine serum, 25 mM NaHCO3, 5 mM HEPES, penicillin (50 U/ml), and streptomycin (50 g/ml). The cells were maintained as subconfluent monolayers and were used between passages [5][6][7][8][9][10][11][12][13][14][15]. Both cell lines were maintained at 37°C in an atmosphere of 5% CO2 in air.…”