| Research Abstract |
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.
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