Mayne K M, Yoshii K, Yu L, Lester H A, Davidson N
Division of Biology, California Institute of Technology, Pasadena 91125.
Brain Res. 1987 Sep;388(3):191-7. doi: 10.1016/0169-328x(87)90026-x.
In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [125I]alpha-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the gamma subunit from Torpedo was replaced by the homologous region of the delta subunit from mouse. None of the chimeric subunits was able to replace the Torpedo gamma, mouse delta, or Torpedo delta subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor.
在本研究中，将体外合成的编码小鼠和电鳐烟碱型乙酰胆碱受体亚基的mRNA注射到非洲爪蟾卵母细胞中，随后进行测定以检测其在卵母细胞表面的组装情况（使用[125I]α-银环蛇毒素结合法）以及乙酰胆碱诱导的电导（使用电压钳法）。我们通过注射4种亚基特异性mRNA的所有8种可能组合，在非洲爪蟾卵母细胞中构建了杂合乙酰胆碱受体，其中单个亚基来自另一个物种。对于每种杂合组合，均可检测到组装和电导情况。我们还构建了编码嵌合乙酰胆碱受体亚基的cDNA克隆，其中电鳐γ亚基的一部分被小鼠δ亚基的同源区域所取代。就组装或功能而言，没有一个嵌合亚基能够替代电鳐γ、小鼠δ或电鳐δ亚基。因此，我们得出结论，蛋白质链中相距较远（且未知）的部分对于亚基间相互作用是必需的，而这种相互作用最终导致受体的功能性组装。
