ENHANCEMENT OF RECOMBINANT ALPHA-1-BETA-1-GAMMA-2L GAMMA-AMINOBUTYRICACID(A) RECEPTOR WHOLE-CELL CURRENTS BY PROTEIN-KINASE-C IS MEDIATED THROUGH PHOSPHORYLATION OF BOTH BETA-1 AND GAMMA-2L SUBUNITS

The gamma-aminobutyric acid(A) (GABA)(A) receptor (GABAR) beta 1 and g amma 2L subtypes have been shown to be phosphorylated in vitro by prot ein kinase C (PKC) [J. Biol. Chem. 267:14470-14476 (1992); Neuron 12:1 081-1095 (1994)]. To determine the physiological consequences of phosp horylation of GABAR isoforms containing the beta(1) and gamma 2L subty pes, the specific serine residues phosphorylated by PKC (beta 1 S409, gamma 2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type (alpha 1 beta 1 gamma 2L GABARs) and three mut ant GABAR isoforms [alpha 1 beta 1 gamma 2L(S327A, S343A), alpha 1 bet a 1(S409A)gamma 2L, and alpha 1 beta 1(S409A)gamma 2L(S327A, S343A) GA BARs) were expressed in mouse L929 fibroblasts through transient cotra nsfection. Recordings were obtained from each cell with the use of the whole-cell patch-clamp technique. The initial recording was made with the use of control intrapipette solution, and a second recording from the same cell was obtained with pipettes containing either constituti vely active PKC [protein kinase M (PKM)] or control solution to obtain paired GABA concentration-response relationships. All GABAR isoforms studied had equivalent maximal GABA currents and similar GABA concentr ation-response profiles under the control condition. Intracellular PKM treatment increased the maximal current and EC(50) value in cells exp ressing wild-type GABARs. However, PKM reimpalement did not significan tly change these parameters in cells expressing any of the mutant GABA R isoforms, indicating that the mutation of either the beta 1 or gamma 2L subtype alone was sufficient to prevent enhancement of GABAR curre nt by PKM. No significant changes were obtained during control reimpal ement recordings of wild-type or mutant receptors. Furthermore, PKM tr eatment did not alter the time constants of GABA current desensitizati on kinetics measured from cells expressing wildtype or mutant receptor s. These data thus suggest that PKC phosphorylation of the beta 1 and gamma 2L subtypes enhances GABAR current and that both subtypes are re quired for complete PKC-mediated enhancement of alpha 1 beta 1 gamma 2 L GABAR current.