Journal of Molecular Biology
Volume 342, Issue 1, 3 September 2004, Pages 333-343
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Trimeric Architecture of Homomeric P2X2 and Heteromeric P2X1+2 Receptor Subtypes

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Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.

Introduction

Ligand-gated ion channels (LGICs) serve to communicate chemical information rapidly across membranes. They achieve this by transducing the binding of extracellular ligands into a conformational change that results in the opening of an intrinsic transmembrane ion channel pore to allow for the flow of selected ions along their electrochemical gradient. On the basis of their amino acid sequences and membrane threading patterns, LGICs have been grouped into three major classes:1, 2 (i) the nicotinic acetylcholine receptor (nAChR) superfamily embracing the ionotropic receptors for acetylcholine, serotonin, glycine, and GABA (γ-aminobutyric acid); (ii) the cationic glutamate receptor (iGluR) family including AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid), NMDA (N-methyl-d-aspartate), and kainate receptors; and (iii) the ATP-gated P2X receptor family. The three LGIC classes do not share sequence homology. They possess, however, the same basic structural elements to fulfil their function such as large ligand-binding extracellular domains and smaller intracellular domains on either side of the membrane linked by transmembrane domains, some of which line the pore. Moreover, LGICs are organized by symmetric or pseudosymmetric arrangements of several subunits like their cousins, the voltage-gated channels.

To date, no 3D crystal structure of an intact member of one of the three major LGIC classes has been reported. The class I LGIC that is structurally best characterized is the nAChR at neuromuscular junctions. It consists of a pentameric barrel stave-like array of homologous subunits arranged in a circular order around a central ion channel. The overall shape and structural changes associated with nAChR activation have been visualized at 4.6 Å resolution by electron microscopy.3 Atomic details of the ligand-binding domain have been revealed by the solved crystal structure of a soluble snail acetylcholine-binding protein that is homologous to the extracellular domain of the nAChR.4

iGluRs were first thought to share the pentameric architecture of the nAChRs. However, both biochemical and electrophysiological data favor the view that iGluRs form tetramers similar to K+ channels (for references see Dingledine et al.5) Detailed structural information of the ligand-binding core is available from the crystal structure of a soluble fusion protein comprising two extracellular regions, which represent ∼25% of the molecular mass of the complete subunit.6 The ligand-binding cores tend to crystallize as dimers,7 suggesting that the assembled tetramer represents a dimer-of-dimers.

P2X receptors open in response to extracellular ATP released from neuronal and non-neuronal cells8 an intrinsic channel with almost equal permeability to Na+/K+ and a relatively high permeability to Ca2+. All P2X subunits share a common membrane topology with cytosolic N and C termini, two membrane-spanning hydrophobic domains (M1 and M2), and a large intervening hydrophilic extracellular loop. On the basis of cysteine scanning mutagenesis, both M19 and M210, 11 have been shown to contribute to the ion permeation pathway. Given that P2X subunits possess two transmembrane domains like inward rectifying K+ channels, a tetrameric organization was anticipated. However, biochemical analyses of recombinant P2X1 and P2X3 receptors revealed an unexpected trimeric subunit organization12 that is consistent with functional studies.13, 14

Incorrect architectures have been initially assigned not only to iGluRs, but also to the bacterial mechanosensitive channel mscL, which was first determined by chemical cross-linking to be a hexamer, and later found by X-ray crystallography to be a pentamer.15 It is not surprising therefore that concern about the validity of this unusual trimeric architecture of P2X receptors has been expressed,16 and indeed, kinetic data have been reported that imply a tetrameric organization of P2X2 receptors.17 The aim of this study was to carefully re-evaluate biochemically the assembly properties and oligomeric state of slowly desensitizing homomeric P2X receptors by focusing in particular on P2X2 homomers and P2X1+2 heteromers. P2X6 subunits were also studied because of their known inability to form functional homomeric receptors in Xenopus oocytes. In addition to blue native PAGE analysis, we used, as a novel independent approach, selective cell surface radioiodination followed by chemical cross-linking of plasma membrane-bound P2X receptors. We show that both non-desensitizing homomeric and heteromeric P2X receptors share a trimeric subunit organization with the previously described desensitising P2X1 and P2X3 receptors.12

Section snippets

Intracellular rat and human P2X2 subunits exhibit distinct assembly states

The rP2X2 receptor, originally cloned from PC12 rat phaeochromocytoma cells,18 is expressed in a variety of neurons in the peripheral and central nervous system, but also pancreas, cochlea, bone, and cardiac muscle.16 The term P2X2 distinguishes the non-desensitizing full length P2X2 subunit from the desensitizing splice variant termed P2X2B, which lacks a stretch of 69 amino acid residues C-terminal to M2.19

In contrast to rP2X1 and rP2X3 subunits,12 the rP2X2 protein did not migrate as a

Discussion

The present study extends previous findings by showing that in addition to homomeric P2X1 and P2X3 receptors, natively purified P2X2, P2X4 and P2X5 receptors and heteromeric rP2X1+2 receptors are rather stable non-covalent assemblies of three subunits. This evaluation relies on a comparison of non-denatured and partially denatured P2X receptors on blue native PAGE gels, and, in addition, for the first time, on chemical cross-linking of plasma membrane-bound P2X receptors in the natural lipid

LGIC cDNA constructs

All LGIC cDNAs used here were subcloned into vector pNKS2, which contains a long poly(A) tract for efficient translation in Xenopus oocytes.40 In addition, all the LGIC cDNA constructs were endowed with virtually the same 50 nucleotides long 5′ non-translated sequence between the SP6 polymerase-binding site and the initiating ATG corresponding to an optimized sequence that has been shown to support maximal protein synthesis in the rabbit reticulocyte system.41 Cloned cDNAs, insertions and

Acknowledgements

We thank Dr Florentina Soto for providing the rP2X5 and rP2X6 clones. Also, we thank Drs Annette Nicke and Bruce A. Cunningham for critical reading and helpful comments on the manuscript. This work was supported by grants of the Deutsche Forschungsgemeinschaft to G.S. (Schm 536/2-3, 536/2-4, Schm 536/6-1, and GRK 137/2). We dedicate this paper to Professor Heinrich Betz on the occasion of his 60th birthday.

References (50)

  • M. Garcia-Guzman et al.

    Molecular cloning and functional expression of a novel rat heart P2X purinoceptor

    FEBS Letters

    (1996)
  • C. Büttner et al.

    Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors

    J. Biol. Chem.

    (2001)
  • J. Rettinger et al.

    Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor

    J. Biol. Chem.

    (2000)
  • H. Schägger et al.

    Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form

    Anal. Biochem.

    (1991)
  • H. Schägger et al.

    Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis

    Anal. Biochem.

    (1994)
  • N. Le Novere et al.

    LGICdb: the ligand-gated ion channel database

    Nucl. Acids Res.

    (2001)
  • R.A. North

    P2X receptors: a third major class of ligand-gated ion channels

    Ciba Found. Symp.

    (1996)
  • K. Brejc et al.

    Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors

    Nature

    (2001)
  • R. Dingledine et al.

    The glutamate receptor ion channels

    Pharmacol. Rev.

    (1999)
  • E. Gouaux

    Structure and function of AMPA receptors

    J. Physiol.

    (2004)
  • Y. Sun et al.

    Mechanism of glutamate receptor desensitization

    Nature

    (2002)
  • P. Bodin et al.

    Purinergic signalling: ATP release

    Neurochem. Res.

    (2001)
  • W.R. Haines et al.

    On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor

    J. Neurosci.

    (2001)
  • F. Rassendren et al.

    Identification of amino acid residues contributing to the pore of a P2X receptor

    EMBO J.

    (1997)
  • T.M. Egan et al.

    A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method

    J. Neurosci.

    (1998)
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    Present addresses: A. Aschrafi, Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; J. Rettinger, Max Planck Institute of Biophysics, Marie-Curie-Strasse 13-15, D-60439 Frankfurt am Main, Germany.

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