Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes
Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes
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磷酸化在爪蟾卵母细胞表达的乙酰胆碱受体脱敏中的作用
DOI：
10.1523/jneurosci.14-07-04185.1994
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
R. Huganir
中科院分区：
文献类型：
--
作者：
PW Hoffman;A. Ravindran;R. Huganir
烟碱型乙酰胆碱受体（AChR）是由化学计量学为α 2 β γ δ的四种类型的亚基组成的五聚体复合物。这些亚基已被cAMP依赖性蛋白激酶（PKA）蛋白激酶C和蛋白酪氨酸激酶差异磷酸化。各种研究表明，AChR在体外和体内的磷酸化调节受体的脱敏率。在这项研究中，我们已经使用了位点特异性突变和膜片钳技术，以检查磷酸化的作用，在调节脱敏的非洲爪蟾卵母细胞中表达的AChR的野生型AChR的非洲爪蟾卵母细胞的表达结果在组成性磷酸化的AChR的γ和δ亚基。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。毛喉素或cAMP类似物或与PKA的催化亚基共表达，卵母细胞中的AChR的磷酸化没有显着增强，这表明卵母细胞中PKA的基础活性足以使受体磷酸化到高化学计量。使用位点特异性诱变，磷酸化位点被确定为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来测量使用快速灌注系统的ACh的重复脉冲的外向补丁的响应。野生型和突变型受体表现出快速的浓度依赖性激活和脱敏应用激动剂。整体平均电流的脱敏的时间常数范围从几百毫秒在低ACh浓度到100-200毫秒在饱和浓度。突变型受体缺乏所有磷酸化位点的脱敏时间常数显着慢于野生型磷酸化受体在所有浓度的乙酰胆碱测试。此外，突变受体的丝氨酸残基改变为谷氨酸残基，以模仿磷酸化丝氨酸残基的负电荷产生的受体，其脱敏率接近野生型磷酸化受体。这些结果进一步支持烟碱乙酰胆碱受体的磷酸化调节脱敏速率。
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.
用加州鱼雷乙酰胆碱受体重建的脂蛋白体的特性。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Huganir,RL;Racker,E
通讯作者：
Racker,E
Forskolin 通过独立于腺苷酸环化酶激活的机制改变乙酰胆碱受体门控。
影响因子：
3.6
作者：
White,MM
通讯作者：
White,MM