The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity.
P2X(7) 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, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) 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. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) 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 P2X(7) 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 P2X(7) channel gating sensitive to intracellular regulation.
Becker
Daniel
D
Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale and the.
Woltersdorf
Ronja
R
Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule Aachen (RWTH), Aachen University, Wendlingweg 2, D-52074 Aachen, Germany.
Boldt
Wolfgang
W
Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale and the.
Schmitz
Stephan
S
Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule Aachen (RWTH), Aachen University, Wendlingweg 2, D-52074 Aachen, Germany.
Braam
Ursula
U
Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule Aachen (RWTH), Aachen University, Wendlingweg 2, D-52074 Aachen, Germany.
Schmalzing
Günther
G
Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule Aachen (RWTH), Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: gschmalzing@ukaachen.de.
Markwardt
Fritz
F
Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale and the. Electronic address: Fritz.Markwardt@medizin.uni-halle.de.
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058486:Receptors, Purinergic P2X7; D014982:Xenopus laevis / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate