Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry α₂βγδ. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes. Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the γ and δ subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the γ subunit and serines 361 and 362 on the δ subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of outside-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild- type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates its rate of desensitization.