Regulation of acetylcholine receptor phosphorylation by calcium and calmodulin

Acetylcholine receptor-enriched membranes prepared from frozen electric organ of Torpedo californica by differential centrifugation and density step gradient centrifugation were assayed for endogenous phosphorylation in the absence and presence of calmodulin and calcium. Each of the membrane fractions exhibited a 3- to 6-fold stimulation of endogenous phosphorylation by calcium and calmodulin. Both calcium and calmodulin were needed for maximal stimulation although calcium alone afforded a small, reproducible stimulation of endogenous phosphorylation. In the presence of fluoride, a phosphatase inhibitor, the calmodulin plus calcium stimulation was increased an additional 3-fold. The phosphorylation reaction was rapid, and maximal phosphorylation was achieved in 2 min. Stimulation of phosphorylation by calcium and calmodulin was completely inhibited by 25 microM trifluoperazine; at 50 microM it inhibited basal phosphorylation by 60%, suggesting that most of the basal phosphorylation may be due to the endogenous calmodulin present in our membrane preparation. NaDodSO4/polyacrylamide gel electrophoresis revealed that at least three of the phosphorylated species (both in the presence and in the absence of calcium and calmodulin) correspond to subunits of the purified acetylcholine receptor from T. californica (i.e., 65,000, 58,000, and 50,000 daltons) which are the beta, gamma, and delta subunits of the receptor.