CA2-DEPENDENT PROTEIN KINASE-II AND PROTEIN-KINASE-C PHOSPHORYLATE A SYNTHETIC PEPTIDE CORRESPONDING TO A SEQUENCE THAT IS SPECIFIC FOR THEGAMMA-2L SUBUNIT OF THE GABA(A) RECEPTOR( CALMODULIN)

The gamma2 subunit of the GABA(A) receptor (GABA(A)-R) is alternativel y spliced. The long variant (gamma2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylati on. Previous studies have demonstrated that a peptide or fusion protei n containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulat ed phosphorylation. We have examined the ability of PKA, PKC, and Ca2/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracell ular loop of the gamma2L subunit and inclusive of the alternatively sp liced phosphorylation consensus sequence site. PKC and CAM kinase II p roduced significant phosphorylation of this peptide, but PKA was ineff ective. The K(m) values for PKC- and CAM kinase II-stimulated phosphor ylation of this peptide were 102 and 35 muM, respectively. Maximal vel ocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC a nd CAM kinase II, respectively. The phosphorylation site in the eight- amino-acid insert of the gamma2L subunit has been shown to be necessar y for ethanol potentiation of the GABA(A)-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of et hanol on GABAergic function.