Department of Physiology, Anatomy and Genetics, University of Oxford.
Department of Physiology, Anatomy and Genetics, University of Oxford;
J Vis Exp. 2021 Mar 11(169). doi: 10.3791/61401.
We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.
我们开发了一种在细胞或膜环境中测量腺嘌呤核苷酸与完整功能跨膜受体结合的方法。该方法结合了带有荧光非典型氨基酸 ANAP 标记的蛋白质的表达，以及 ANAP 与荧光（三硝基苯）核苷酸衍生物之间的 FRET。我们展示了在电压钳下在未覆盖的质膜和分离的、内向外膜片中测量到的与 ANAP 标记的 KATP 离子通道结合的核苷酸的实例。后者允许同时测量配体结合和通道电流，这是蛋白质功能的直接读数。我们广泛讨论了数据处理和分析，以及潜在的陷阱和伪影。该方法为研究 KATP 通道的配体依赖性门控提供了丰富的机制见解，并且可以很容易地适应于研究其他核苷酸调节蛋白或任何可以识别合适荧光配体的受体。
