ROLE OF PHOSPHORYLATION IN DESENSITIZATION OF ACETYLCHOLINE-RECEPTORSEXPRESSED IN XENOPUS-OOCYTES

The nicotinic acetylcholine receptor (AChR) is a pentameric complex ma de up of four types of subunits in the stoichiometry alpha(2) beta gam ma delta. These subunits have been shown to be differentially phosphor ylated by cAMP-dependent protein kinase (PKA), protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that pho sphorylation of the AChR in vitro and in vivo regulates the rate of de sensitization of the receptor. In this study we have used site-specifi c mutagenesis and patch-clamp techniques to examine the role of phosph orylation in the regulation of desensitization of the AChR expressed i n Xenopus oocytes. Expression of wild-type AChR in Xenopus oocytes res ults in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high bas al level of PKA in oocytes since a specific peptide inhibitor of PKA c ompletely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significant ly enhanced by forskolin or cAMP analogs or by coexpression with the c atalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichio metry. Using site-specific mutagenesis, the sites of phosphorylation w ere determined to be serines 353 and 354 on the gamma subunit and seri nes 361 and 362 on the delta subunit. To examine the functional proper ties 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. W ild-type and mutant receptors showed rapid concentration-dependent act ivation and desensitization to applied agonist. The time constant of d esensitization of ensemble mean currents ranged from several hundred m illiseconds at low ACh concentrations to 100-200 msec at saturating co ncentrations. 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 glu tamate residues in order to mimic the negative charge of the phosphory lated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These res ults provide further support that phosphorylation of the nicotinic ACh receptor regulates its rate of desensitization.