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 beta-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 microm. This current was totally blocked by the combined application of NO 711 (5 microm) and beta-alanine (50 microm). We obtained an EC(50) of 5 microm 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 microm 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 microm) 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.
Garduño
Julieta
J
Department of Physiology, Biophysics, and Neuroscience, Centro de Investigación y de Estudios Avanzados, 07360 Mexico City, Mexico.
Elenes
Sergio
S
Cebada
Jorge
J
Becerra
Elizabeth
E
García
Ubaldo
U
D000818:Animals; D003400:Astacoidea; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D002712:Chlorides / Q000378:metabolism; D005260:Female; D018756:GABA Antagonists / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D006458:Hemolymph / Q000737:chemistry; D007150:Immunohistochemistry; D017136:Ion Transport / Q000187:drug effects; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D026901:Membrane Transport Proteins; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D002712:Chlorides; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D012964:Sodium