P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity

Background and Purpose P2X receptors are trimeric ligand‐gated ion channels that open a cation‐selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. Experimental Approach Here, we examine the impact of P2X2 receptor inter‐subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. Key Results We demonstrate that predicted extracellular domain inter‐subunit interfaces display a higher‐than‐expected density of missense variations and that the majority of mutations that disrupt putative inter‐subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. Conclusion and Implications We provide the first structural mapping of the mutational distribution across the human population in a ligand‐gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand‐gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.