Molecular Cloning of a GABA Receptor Subunit from Crayfish and Voltage-Clamp Analysis of Homo-Oligomeric Receptor Expressed in Hek Cells

In vertebrate central nervous system, GABA acts as inhibitory neurotransmitter by binding to two general classes of receptors: chloride ligand-gated ion channels complex GABAARs and metabotropic GABABRs that are linked via trimeric G-proteins to potassium channels. However, in invertebrates GABA can activate inward cationic currents through an homo-oligomeric receptor as has been described in the nematode C. elegans, where the central ionic conduction pore corresponds to an amino acidic sequence that permits the cationic flux. Another kind of cation-selective heteromultimeric receptor formed by the combination of two ligand-gated channel subunits (LCCH3 and GRD) was described in the fruit fly D. melanogaster. Similarly, in the x-organ neurons of the crayfish Procambarus clarkii we have identified two GABA-gated currents: an early transient inward current depending on the extracellular sodium concentration, and other one generated by chloride ions. Both currents are activated by muscimol and blocked by picrotoxin, while cis-aminocrotonic acid only activates the chloride current. A cDNA encoding an ionotropic GABAR subunit was isolated from these neurons and transiently transfected into HEK 239T cells. Pharmacologically this subunit forms an anionic ligand-gated GABAR activated by muscimol and cis-aminocrotonic acid, and blocked by picrotoxin but not by bicuculline. Currently we are conducting experiments in our laboratory aimed to identify the cDNA that encodes for the subunit that forms the cationic receptor.Supported by CONACYT scholarship 315151 to ENJ-V and grant 131778 to JMA.