Abstract
P2X receptors (P2XRs) are non-selective cation channels that open in response to ATP binding and mediate crucial physiological roles throughout the human body. Despite their physiological relevance, much of the molecular pharmacology of P2XRs remains unclear. We therefore aim to define the precise physico-chemical contribution of side chains in ATP binding. We combined electrophysiological techniques and conventional mutagenesis on P2X2Rs expressed in Xenopus laevis oocytes together with a novel approach based on the insertion of unnatural amino acids (UAAs). Preliminary studies focused on conventional mutagenesis and insertion of UAAs at the P2X2R ATP binding site. Replacement of conserved lysine with arginine and glutamine suggests that under physiological condition some residues need to be positively charged in order to coordinate ATP, while others may not. The data further underline a previously underestimated contribution of hydrophobic amino acids for ATP recognition. To further investigate the role of conserved positively charged amino acid we aimed to incorporate unnatural amino acid. Initial studies demonstrate that subtle lysine analogues can be successfully incorporated into P2X2R and pave the way for atomic-scale manipulations of the ATP binding site in P2XRs.Overall, these data shed new light on the importance of both hydrophobic and positively charged amino acid in ATP coordination.
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