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Journal of Neuroscience, Vol 14, 4185-4195, Copyright © 1994 by Society for Neuroscience
Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes
PW Hoffman, A Ravindran and RL Huganir
Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up
of four types of subunits in the stoichiometry alpha 2 beta gamma delta.
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 gamma and delta 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 gamma subunit and serines 361 and 362 on the delta 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 out-side-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 rate of desensitization.
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