The P2X7 Carboxyl Tail Is a Regulatory Module of P2X7 Receptor Channel Activity

• Published in: The Journal of biological chemistry 2008-09, Vol.283 (37), p.25725-25734
• Main Authors: Becker, Daniel, Woltersdorf, Ronja, Boldt, Wolfgang, Schmitz, Stephan, Braam, Ursula, Schmalzing, Günther, Markwardt, Fritz
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Animals; Cell Membrane - metabolism; Cross-Linking Reagents - pharmacology; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Biological; Oocytes - metabolism; Patch-Clamp Techniques; Protein Structure, Tertiary; Receptors, Purinergic P2 - chemistry; Receptors, Purinergic P2X7; Xenopus laevis - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M803855200

P2X7 receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X7 subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X7 subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X7 channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only ∼5% of the full-length P2X7 receptor current. Co-assembly of channel core subunits with full-length P2X7 subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X7 receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X7 channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X7 receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X7 channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X7 subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X7 channel gating sensitive to intracellular regulation.