Spectroscopic and x-ray scattering studies of the interaction of a volatile general anesthetic with ion channel proteins

Ion channels play crucial roles in nervous system activities. The significance of ion channel functions has been known for decades. However, the mechanisms behind the function or how an ion channel function is affected by drug molecules are not well understood. This study aims to develop new approaches which facilitate uncovering the nature of general anesthetic functions and the mechanism of the voltage-gated potassium channel (Kv) function.;Firstly, a synthetic ion channel protein was designed to investigate the nature of volatile general anesthetic function. A non-biological amino acid, cyano-phenylalanine, was employed to probe the interaction between the anesthetic molecule and the synthetic protein. The application of cyano-phenylalanine enables simultaneously applying fluorescence and infrared spectroscopy techniques to study this interaction. Molecular dynamics simulations performed via collaboration were required to interpret the infrared results and showed that the anesthetic interacted with protein backbone atoms exposed in the cavity, thereby modifying the local environment of the probe. Secondly, the distribution of general anesthetic halothane along the length of the synthetic protein bundle was investigated via X-ray reflectivity. Comparison of the halothane distribution along the target protein bundle, which has a designed binding site, with the distribution along a control protein bundle, in which the binding site is removed, reveals that halothane preferentially associates within the designed binding cavity. The results from the synthetic ion channel protein justify the direct mechanism, in which general anesthetic molecules affect the function of their natural targets, namely the ligand-gated ion channels, by directly binding to the cavities within the channels. Thirdly, the structure of the voltage-sensitive domain (VSD) of a Kv channel on a solid substrate was studied. Two fundamentally different approaches for vectorially immobilizing the VSD were developed. The formation of the protein monolayer and the vectorial-orientation of the protein molecules were investigated via interferornetric X-ray reflectivity. The electron density profile of the tethered protein monolayer is consistent with the profile computed from the crystal structure, irrespective of the preparation procedure. This work paves the way for employing time-resolved techniques to investigate the nature of the VSD as well as the intact Kv channel function.