PHOSPHORYLATION OF THE GABA(A) RECEPTOR GAMMA-2L SUBUNIT IN RAT SENSORY NEURONS MAY NOT BE NECESSARY FOR ETHANOL SENSITIVITY

The effect of ethanol on the current activated by 2.5 to 40 mu M gamma -aminobutyric acid (GABA) was studied in freshly isolated rat dorsal r oot ganglion (DRG) neurons under voltage clamp in the whole-cell and p erforated-patch recording configurations. Our results confirmed that G ABA(A)-activated current in these neurons was insensitive to ethanol a t concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Wei ght, Ethanol inhibits NMDA-activated current but does not alter GABA-a ctivated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332-336.]. In addition, the ethanol sensitivity of GABA recept ors was studied under conditions that promote phosphorylation of the P KC site on the gamma 2L subunit. The presence of the gamma 2L and othe r subunit mRNAs was detected by reverse transcription (RT) of total RN A purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response re mained insensitive to 2.5-100 mM ethanol despite: (i) the extracellula r preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (P MA); (ii) raising free intracellular Ca2+ ([Ca2+](i)) from 7 to 100 or 600 nM by altering the intracellular Ca2+/EGTA ratio; (iii) intracell ular application of PKC (0.247 U ml(-1)); and (iv) combining the intra cellular application of 1 mu M okadaic acid and 30 mu M peptide 3 with the extracellular application of 20 nM PMA. These results suggest tha t phosphorylation of the gamma 2L subunit is not the only requirement for ethanol sensitivity of GABA(A) receptors. (C) 1998 Elsevier Scienc e B.V. All rights reserved.