TY - JOUR

T1 - P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity

AU - Gasparri, Federica

AU - Sarkar, Debayan

AU - Bielickaite, Sarune

AU - Poulsen, Mette Homann

AU - Hauser, Alexander Sebastian

AU - Pless, Stephan Alexander

N1 - Themed Issue: Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary).

PY - 2022

Y1 - 2022

N2 - Background and PurposeP2X 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 ApproachHere, 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 ResultsWe 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 ImplicationsWe 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

AB - Background and PurposeP2X 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 ApproachHere, 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 ResultsWe 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 ImplicationsWe 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

KW - agonist sensitivity

KW - genetic variation

KW - ion channel gating

KW - missense mutations

KW - purinergic receptor

KW - receptor modulation

U2 - 10.1111/bph.15830

DO - 10.1111/bph.15830

M3 - Journal article

C2 - 35285517

VL - 179

SP - 3859

EP - 3874

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

IS - 14

ER -