The ‘y-aminobutyric acidA (GABA) A receptor(GABAR)/31 and y2L subtypes have been shown to be phosphorylated in vitro by protein kinase C (PKC)[J. Biol. Chem. 267: 14470-14476 (1 992); Neuron 12: 1 081-1 095 (1994)]. To determine the physiological consequences of phosphorylation of GABAR isoforms containing the/31 and-y2L subtypes, the specific serine resi-dues phosphorylated by PKC (/31 S409,‘y2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type(al/31-y2L GABARs) and three mutant GABAR iso-forms(al/31 y2L (S327A, S343A), al/31 (S409A)’y2L, and al/31 (S409A)’y2L (S327A, S343A) GABARs) were expressed in mouse L929 fibroblasts through transient cotransfection. 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 contaming either constitutively active PKC [protein kinase M (PKM)] or control solution to obtain paired GABA concentration-re-sponse relationships. All GABAR isoforms studied had equiva-lent maximal GABA currents and similar GABA concentration-response profiles under the control condition. Intracellular PKM treatment increased the maximal current and EC50 value in cells expressing wild-type GABARs. However, PKM reimpalement did not significantly change these parameters in cells express-ing any of the mutant GABAR isoforms, indicating that the mutation of either the/31 or-y2L subtype alone was sufficient to prevent enhancement of GABAR current by PKM. No signifi-cant changes were obtained during control reimpalement re-cordings of wild-type or mutant receptors. Furthermore, PKM treatment did not alter the time constants of GABA current desensitization kinetics measured from cells expressing wild-type or mutant receptors. These data thus suggest that PKC phosphorylation of the/31 and-y2L subtypes enhances GABAR current and that both subtypes are required for complete PKC-mediated enhancement of al/31 y2L GABAR current.

The GABA. R is a ligand-gated Cl channel that is distributed widely throughout the central nervous system (1). Different subunit families of GABAR(a,/3, y, 6, p) and multiple subtypes of each family (al-6,/31-4, yl-4, pl-2) have been cloned (2, 3). Each subtype is thought to contain an aminoterminal extracellular domain, four membrane spanning domains, and a large and highly variable cytoplasmic domain between the third and fourth transmembrane domains, and GABARS are thought to be composed of combinations of five subunit subtypes(4). The GABAR is regulated by a number