Studies on receptor structure-function relationship using molecular biological techniques and GFP molecular visualization

为了阐明ATP激活的受体/通道(P2X受体)的结构和功能之间的关系，人们利用分子生物学技术引入突变(氨基酸替换)。结合GFP分子可视化技术验证突变体的表达。先前的研究表明，当Gly247被丙氨酸取代时，P2X2受体的活性就会丧失。该区域被认为在拓扑上类似于tRNA氨基酰基合成酶的ATP结合域。在这些酶中，芳香氨基酸残基被认为有助于识别ATP分子的腺嘌呤部分。因此，两个高度保守的芳香族氨基酸残基(Phe240和Trp256)被非芳香族残基取代。通过这些替换获得的电生理数据表明，Trp256是通道活动所必需的。酪氨酸部分补偿了Trp256，但包括苯丙氨酸在内的其他氨基酸没有补偿。通过GFP连接受体的荧光图像和膜组分的免疫印迹分析，证实了非功能突变体在细胞表面的表达。结果表明，Trp256的替换不影响蛋白质的翻译或运输过程，但影响细胞膜上受体蛋白的结构。通过这些研究，将分子生物学技术与GFP分子可视化相结合，对于阐明受体的结构和功能关系具有重要价值。此外，引入中性氨基酸的研究表明，333位氨基酸以立体方式阻止钙离子进入通道孔。研究还表明，细胞内的二硫键影响对三磷酸腺苷的反应。

To clarify the relationship between the structure and the function of ATP-activated receptor/channel (P2X receptor), mutations (amino acid substitutions) were introduced by molecular biological techniques. GFP molecular visualization was combined to verify the expression of the mutants. Previous studies had shown that the activity of P2X2 receptor is lost when Gly247 was replaced with alanine. This region is assumed to be topologically similar to the ATP-binding domain of tRNA amino acyl synthetases. In these enzymes, aromatic amino acid residues are believed to contribute to the recognition of the adenine moiety of ATP molecules. Thus, two highly conserved aromatic amino acid residues (Phe240 and Trp256) were replaced with non-aromatic residues. The electrophysiological data obtained with these replacements showed that Trp256 is indispensable for the channel activity. Tyrosine partly compensated for Trp256, but other amino acids including phenylalanine failed. The expression of the non-functional mutants on cell surface was confirmed by the fluorescent images of GFP-connected receptors and the immunoblotting analysis of membrane fractions. The results suggested that the replacement of Trp256 does not affect protein translation or trafficking processes, but affects the structure of the receptor protein in cell membrane. Through these studies, the combination of molecular biological techniques and GFP molecular visualization is valuable for the clarification of the receptor structure and function relationship. In addition, the study with neutral amino acid introduction has shown that the amino acid at the position 333 sterically prevents calcium ion approach to the channel pore. It has been also shown that an intracellular disulfide bond affects the responsiveness to ATP.