Molecular cloning, functional characterization and differential expression of two novel GABAAR-like subunits from red swamp crayfish Procambarus clarkii

Abstract

In this work, we cloned and functionally expressed two novel GABA_A receptor subunits from Procambarus clarkii crayfish. These two new subunits, PcGABA_A-α and PcGABA_A-β2, revealed significant sequence homology with the PcGABA_A-β subunit, previously identified in our laboratory. In addition, PcGABA_A-α subunit also shared a significant degree of identity with the Drosophila melanogaster genes DmGRD (GABA and glycine-like receptor subunits of Drosophila) as well as PcGABA_A-β2 subunit with DmLCCH3 (ligand-gated chloride channel homolog 3). Electrophysiological recordings showed that the expression in HEK cells of the novel subunits, either alone or in combination, failed to form functional homo- or heteromeric receptors. However, the co-expression of PcGABA_A-α with PcGABA_A-β evoked sodium- or chloride-dependent currents that accurately reproduced the time course of the GABA-evoked currents in the X-organ neurons from crayfish, suggesting that these GABA subunits combine to form two types of GABA receptors, one with cationic selectivity filter and the other preferentially permeates anions. On the other hand, PcGABA_A-β2 and PcGABA_A-β co-expression generated a chloride current that does not show desensitization. Muscimol reproduced the time course of GABA-evoked currents in all functional receptors, and picrotoxin blocked these currents; bicuculline did not block any of the recorded currents. Reverse transcription polymerae chain reaction (RT-PCR) amplifications and FISH revealed that PcGABA_A-α and PcGABA_A-β2 are predominantly expressed in the crayfish nervous system. Altogether, these findings provide the first evidence of a neural GABA-gated cationic channel in the crayfish, increasing our understanding of the role of these new GABA_A receptor subunits in native heteromeric receptors.