Phosphorylation of the GABA A receptor γ2L subunit in rat sensory neurons may not be necessary for ethanol sensitivity

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The effect of ethanol on the current activated by 2.5 to 40 μM γ-aminobutyric acid (GABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons under voltage clamp in the whole-cell and perforated-patch recording configurations. Our results confirmed that GABA A-activated current in these neurons was insensitive to ethanol at concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Weight, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332–336.]. In addition, the ethanol sensitivity of GABA receptors was studied under conditions that promote phosphorylation of the PKC site on the γ2L subunit. The presence of the γ2L and other subunit mRNAs was detected by reverse transcription (RT) of total RNA purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response remained insensitive to 2.5–100 mM ethanol despite: (i) the extracellular preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (PMA); (ii) raising free intracellular Ca 2+ ([Ca 2+] i) from 7 to 100 or 600 nM by altering the intracellular Ca 2+/EGTA ratio; (iii) intracellular application of PKC (0.247 U ml −1 ); and (iv) combining the intracellular application of 1 μM okadaic acid and 30 μM peptide 3 with the extracellular application of 20 nM PMA. These results suggest that phosphorylation of the γ2L subunit is not the only requirement for ethanol sensitivity of GABA A receptors.