Channel gating in the absence of agonist by a homooligomeric molluscan GABA receptor expressed inXenopus oocytes from a cloned cDNA

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Abstract
We have previously described the isolation of a complementary DNA (cDNA) from the freshwater molluscLymnaea stagnalis encoding a polypeptide that exhibits ∼50% identity to the ß-subunits of vertebrate γ-aminobutyric acid (GABA) type A (GABAA) receptor. When expressed inXenopus laevis oocytes fromin vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at −60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of ∼20 pS channels, with an open probability greater than 90% (at a pipette potential of −100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (−100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABAA receptor α1-subunit, but the channels gated by these receptors did not open spontaneously.