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

The gamma2 subunit of the GABA(A) receptor (GABA(A)-R) is alternatively spliced. The long variant (gamma2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated 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 intracellular loop of the gamma2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K(m) values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 muM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma2L subunit has been shown to be necessary 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 ethanol on GABAergic function.