Functional analysis of mutation and partial duplication of α7 nicotinic receptor subunit gene : implication in schizophrenia

1.α-7烟碱受体亚单位基因被认为是精神分裂症的易感基因。在本研究中，我们在一名日本精神分裂症患者中发现了一种新的CHRNA7基因突变。由于突变导致受体胞质环氨基酸替换(G423S)，因此我们研究了野生型(WT)和非洲爪哇卵母细胞表达的α7-G423S突变受体之间的功能差异。在蛋白激酶C激动剂存在的情况下，α7激动剂可诱导α7-G423S突变受体的磷酸化和抑制，但不能抑制WT受体。这些结果表明，G423S突变通过蛋白激酶C依赖的机制促进受体脱敏。CHRNA7基因是部分复制的，并与新基因FAM7A形成杂交(CHRFAM7A)。在本研究中，我们发现CHRFAM7A基因在非洲爪哇卵母细胞和人SH-SY5Y细胞中被翻译成蛋白质(dup-α7)，并且该蛋白质与α7受体亚单位共沉淀。此外，电生理和生化研究表明，Dup-α7蛋白的表达导致α7受体活性降低。这些发现表明存在一种通过dup-α7蛋白调节α7受体活性的新机制。我们检测了两栖动物和海鞘生物碱对神经元型烟碱受体的影响，发现生物碱(+)-205B是α7烟碱受体的专利选择性阻滞剂。我们发现，慢性尼古丁刺激引起下丘脑Gt1-7细胞促性腺激素释放激素通过烟碱受体表达增加，血管平滑肌细胞α7烟碱受体表达上调，提示α7基因G423S突变和DUP-α7蛋白过量扰乱了CHRNA7烟碱受体的功能，是精神分裂症等中枢性胆碱能障碍的易感因素。

1. The α7 nicotinic receptor subunit (CHRNA7) gene has been considered as a susceptible gene for schizophrenia. In this study, we found a novel mutation in CHRNA7 gene in a Japanese schizophrenic patient. Since the mutation results in amino acid substitution (G423S) in the receptor cytoplasmic loop, we investigated functional differences between wild-type (WT) and the α7-G423S mutant receptor expressed in Xenopus oocytes. In the presence of a protein kinase C (PKC) activator, stimulation with α7 agonist induces phosphorylation and inhibition of the α7-G423S mutant receptor, but not WT receptor. These results demonstrate that the G423S mutation promotes the receptor desensitization by a protein kinase C-dependent mechanism.2. The CHRNA7 gene is partially duplicated and forms a hybrid (CHRFAM7A) with a novel gene FAM7A. In this study, we found that the cDNA of CHRFAM7A gene was translated into protein (dup-α7) in Xenopus oocytes and human SH-SY5Y cells, and that the protein was coimmunoprecipitated with α7 receptor subunit. Moreover, electrophysiological and biochemical studies demonstrated that the expression of dup-α7 protein caused the reduction of α7 receptor activity. These findings suggest the existence of a novel mechanism for regulating α7 receptor activity via dup-α7 protein.3. We examined effects of several alkaloids from amphibians and ascidians on neuronal nicotinic receptors, and found that the alkaloid (+)-205B is a patent and selective blocker of α7 nicotinic receptor.4. We found that chronic nicotine stimulation caused enhanced expression of gonadotropin releasing hormone in hypothalamic GT1-7 cells via t nicotinic receptor, and upregulation of α7 nicotinic receptor in vascular smooth muscle cells.These results suggest that the G423S mutation in CHRNA7 and the dup-α7 protein excess perturb the function of α7 nicotinic receptor and serve as predisposing factors for central cholinergic disorders, such as schizophrenia.