Ca²⁺/Calmodulin‐Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ_2L Subunit of the GABA_A Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A‐R) is alternatively spliced. The long variant (γ_2L) 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 γ_2L 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 μM, 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 γ_2L 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.