Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Pharmacology

Background:

• P2X receptors (P2XRs) are ATP-gated ion channels crucial for cellular signaling.
• Desensitization rates vary among P2XR subtypes, with the helical recoil model suggesting cytoplasmic cap stability dictates this process.
• The precise timing of cytoplasmic cap formation in slow-desensitizing P2XRs remains unclear.

Purpose of the Study:

• To elucidate the structural and functional mechanisms underlying P2X4 receptor desensitization.
• To determine the role of the cytoplasmic cap in P2X4 receptor gating and desensitization kinetics.
• To identify potential allosteric binding sites for small-molecule modulator development.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine structures of human P2X4 receptor in multiple functional states.
• Functional assays to assess receptor activity and desensitization kinetics.
• Biochemical analyses to investigate post-translational modifications.

Main Results:

• Cryo-EM structures revealed distinct conformations of P2X4 receptor in apo closed, antagonist-bound inhibited, and ATP-bound desensitized states.
• The cytoplasmic cap is intact in apo closed and inhibited states but absent in the desensitized state, indicating pre-agonist binding formation.
• Lipid interactions stabilize the cytoplasmic cap, modulating desensitization; P2X4 undergoes glycosylation and palmitoylation.
• The antagonist-bound structure highlights an allosteric ligand-binding pocket.

Conclusions:

• The cytoplasmic cap of P2X4 receptors forms prior to ATP binding, preceding desensitization.
• Lipid interactions and post-translational modifications (glycosylation, palmitoylation) are critical for P2X4 receptor function and desensitization.
• The identified allosteric binding site offers a target for developing novel P2X4 receptor modulators.