Hoffman P W, Ravindran A, Huganir R L
Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Neurosci. 1994 Jul;14(7):4185-95. doi: 10.1523/JNEUROSCI.14-07-04185.1994.
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.
烟碱型乙酰胆碱受体（AChR）是一种五聚体复合物，由四种亚基按α2βγδ的化学计量比组成。这些亚基已被证明可被环磷酸腺苷依赖性蛋白激酶（PKA）、蛋白激酶C和一种蛋白酪氨酸激酶进行不同程度的磷酸化。各种研究表明，AChR在体外和体内的磷酸化调节受体的脱敏速率。在本研究中，我们使用位点特异性诱变和膜片钳技术来研究磷酸化在非洲爪蟾卵母细胞中表达的AChR脱敏调节中的作用。野生型AChR在非洲爪蟾卵母细胞中的表达导致AChR的γ和δ亚基发生组成型磷酸化。这种磷酸化显然是由于卵母细胞中PKA的高基础水平，因为PKA的一种特异性肽抑制剂在体外完全消除了卵母细胞提取物对AChR的磷酸化。毛喉素、环磷酸腺苷类似物或与PKA催化亚基共表达均未显著增强卵母细胞中AChR的磷酸化，这表明卵母细胞中PKA的基础活性足以将受体磷酸化至高化学计量比。使用位点特异性诱变，确定磷酸化位点为γ亚基上的丝氨酸353和354以及δ亚基上的丝氨酸361和362。为了研究缺乏磷酸化位点的野生型和突变型受体的功能特性，我们使用膜片钳技术，通过快速灌注系统测量外翻膜片对ACh重复脉冲的反应。野生型和突变型受体对施加的激动剂表现出快速的浓度依赖性激活和脱敏。在低ACh浓度下，总体平均电流脱敏的时间常数范围为几百毫秒，在饱和浓度下为100 - 200毫秒。在所有测试的ACh浓度下，缺乏所有磷酸化位点的突变型受体的脱敏时间常数明显慢于野生型磷酸化受体。此外，将丝氨酸残基突变为谷氨酸残基以模拟磷酸化丝氨酸残基负电荷的突变型受体产生的脱敏速率接近野生型磷酸化受体。这些结果进一步支持了烟碱型乙酰胆碱受体的磷酸化调节脱敏速率。
