Differential phosphorylation of intracellular domains of gamma-aminobutyric acid type A receptor subunits by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase.

y-Aminobutyric acid type A receptor subunits (GABA,) can be divided into five classes, a, p, y, 6, and p, based on sequence homology.W e have used purified fusion proteins of the major intracellular domain of GABA, receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cGMP-dependent protein kinase (PKG) and multifunctional calcium/calmodulin-dependent protein kinase (CAM KII).Both PKG and CAM KII phosphorylated a purified p1 subunit fusion.Both of these kinases phosphorylated serine 409 within the p1 subunit; in addition, CAM KII also phosphorylated serine 384 as determined by site-specific mutagenesis.Fusion proteins of the major intracellular domains of the y,S and yzL subunits were produced.These proteins differ by 8 amino acids (LLRMFSFK).Both the yzL and yzS fusion proteins were excellent substrates of CAM KII.However, the y,L fusion protein was phosphorylated to higher stoichiometry due to the phosphorylation of serine 343 within this 8-amino acid insertion.Both the y,L and yzS subunits were phosphorylated on common residues by CAM KII identified as serine 348 and threonine 350.These results identify specific sites of phosphorylation for CAM KII and PKG within GABA, receptor subunits, suggesting a role for these two kinases in modulating GABA, receptor function in vivo.y-Aminobutyric acid typeA(GABA,)l receptors are the major sites of synaptic inhibition in the brain; they are also the major sites of action of benzodiazepines and barbiturates (1).Molecular cloning has revealed a large diversity of GABA, receptor subunits that can be divided into five subunit classes based on receptor subunits share significant homology to the subunits of both nicotinic acetylcholine and glycine receptors.Members of this superfamily of ligand-gated ion channels are proposed to share a common transmembrane topology consisting of a large N-terminal extracellular domain, four transmembrane do-sequence homology: a(14), P(1-4), y(1-3), 8(1), and P(~-,, (2).GABAA *