Dataset Information

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

ABSTRACT: Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.

SUBMITTER: Salm EJ

PROVIDER: S-EPMC7337098 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

Salm Elizabeth J EJ Dunn Patrick J PJ Shan Lili L Yamasaki Miwako M Malewicz Nathalie M NM Miyazaki Taisuke T Park Joongkyu J Sumioka Akio A Hamer R Richard L RRL He Wei-Wu WW Morimoto-Tomita Megumi M LaMotte Robert H RH Tomita Susumu S

Cell reports 20200601 9

Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modula ...[more]

PMID: 32492420

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Project description:P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

Dataset Information

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

Publications

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

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