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

• Published in: bioRxiv
• Main Authors: Gasparri, Federica, Sarkar, Debayan, Bielickaite, Sarune, Mette Homann Poulsen, Hauser, Alexander, Pless, Stephan Alexander
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 29.01.2022
• Subjects: Channel opening; Inflammation; Ion channels; Ion channels (ligand-gated); Ligands; Mutants; Mutation; Pain perception
• DOI: 10.1101/2021.03.26.436616

Background and Purpose: P2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains. Experimental Approach: Here we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. Key results: We demonstrate that predicted ECD 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. Conclusions and Implications: We provide the first structural mapping of the mutational burden across the human population in a LGIC 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 LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation. Competing Interest Statement The authors have declared no competing interest. Footnotes * Minor text edits