cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor

Abstract

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the γ and δ subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the γ and δ subunits of the receptor. cAMP activation of the endogenous phosphorylation of the γ and δ subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the γ and δ subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of 32 P per mol of receptor, respectively. The initial rates of phosphorylation of the γ and δ subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the γ and δ subunits had K m values of 4.0 and 3.3 μM and V max values of 2.7 and 2.1 μmol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.