Baculovirus Expression Provides Direct Evidence for Heteromeric Assembly of P2X 2 and P2X 3 Receptors

378

149

P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X 1 through P2X 7 ) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homooligomeric complexes, the exception being P2X 6 . When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X 7 . Whereas P2X 1 , P2X 2 , P2X 5 , and P2X 6 were able to assemble with most subunits, P2X 3 and P2X 4 presented a more restricted pattern of coassociation. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP.Investigation of the native receptors mediating extracellular ATP signaling in tissues has been a difficult task due to the lack of useful pharmacological tools. For this reason, the cloning of ATP receptors (the P2 receptor family) and their recombinant expression has proven extremely useful in elucidating the basic properties of these proteins and for providing a template for further study into native P2 receptors. Two families of proteins mediating the actions of ATP have been identified: the metabotropic G protein-coupled P2Y receptors and the ionotropic P2X receptors (1). The P2X receptors are nonselective ion channels thought to be oligomeric in nature, and they are expressed in many excitable and nonexcitable cells, where they mediate a variety of physiological actions, including smooth muscle contractility, neuroendocrine secretion, and modulation of synaptic transmission (2, 3). In addition, recent reports suggest that they may also play an important role in the transmission of pain perception (4, 5). With the molecular identification of seven P2X receptor subunits, our understanding of the biophysical and pharmacological properties of these channels has been considerably increased. However, relatively little is known about the multimeric organization of this new class of channel receptors.Almost all known ionotropic receptors exist as hetero-oligomers (6). Importantly, their functional and pharmacological properties are directly determined by their subunit composition, with different subunit combinations yielding different phenotypes (e...

Section: Resultssupporting

confidence: 89%

Hetero-oligomeric Assembly of P2X Receptor Subunits

Torres

Egan

Voigt

1999

378

149

“…P2X 2/3 hetero-oligomeric channels have been extensively studied (28,29). Based on biochemical and functional evidence, it has been proposed recently that the P2X 2/3 receptor is formed by the head-to-tail association of one P2X 2 and two P2X 3 subunits (9).…”

Section: Discussionmentioning

confidence: 99%

“…3). The ATP analog ␣␤-methylene-ATP (␣␤-me-ATP) was also ineffective in inducing current in HEK cells transfected with P2X 3 (28,29). We studied the trafficking of hetero-oligomeric P2X receptors formed by the association of wild type and trafficking deficient subunits.…”

Section: Specificity Of the Yxxxk Motif And Functional Characterizatimentioning

confidence: 99%

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

Chaumont

Jiang

Penna

et al. 2004

Self Cite

Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD 4 ) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X 2 subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.P2X receptors are extracellular ATP-gated ion channels; they define the third major class of ligand-gated channels together with the glutamate and the nicotinic superfamilies (1). P2X receptors are involved in synaptic transmission in the central and peripheral nervous systems, but in contrast to other ligand-gated channels, they are also widely expressed in peripheral tissues where they participate in physiological processes as diverse as smooth muscle contraction, secretion, and bone resorption. ATP-induced currents have been recorded from a large number of different cell types (2), but the molecular identity of P2X receptors responsible for these currents often remains elusive because of limitations of pharmacological tools to discriminate the different subtypes of P2X channels.Seven P2X subunit genes (P2X 1 -P2X 7 ) are found in the human genome; this number seems to be an accurate estimation of the extent of the P2X family in mammalian species, although in zebrafish two additional subunits appear to exist (2, 3). The basic structural determinants of P2X channels have been established by numerous molecular studies (4). P2X subunits have a membrane topology with two transmembrane domains linked by a large extracellular loop; N and C termini are localized intracellularly (5, 6). To form a channel, P2X subunits are thought to associate as homo-or heterooligomers, composed of three subunits (7, 8) organized in a head-to-tail orientation around a central pore (9). This model is consistent with studies that show that the permeation pathway is associated with transmembrane regions (10 -12), and with the fact that the gating of the channel is in part due to movements of the subunits relative to the each other (12). Conserved charged residues in the extracellular loop have been implicated in ATP binding (13,14), and desensitization is tightly linked to transmembrane domains and intracellular regions lo...

“…These are the P2Y receptors, coupled to G proteins and second messenger pathways, and the P2X receptors, which are ligandgated ion channels (1). Seven subunits of the P2X receptor family (P2X [1][2][3][4][5][6][7] have been characterized at the molecular level (reviewed in Refs. 2 and 3).…”

mentioning

confidence: 99%

“…These show a broad expression pattern compared with other ligand-gated channels, with the various forms being found in central and peripheral nervous system, different types of immune cells, glands, and smooth and skeletal muscles (4). The P2X receptor subunits can form channels as homomultimers or, in some cases, as heteromultimers (5,6). The number of subunits in each channel molecule is not known.…”

mentioning

confidence: 99%

Membrane Topology of an ATP-gated Ion Channel (P2X Receptor)

Newbolt¹,

Stoop²,

Virginio³

et al. 1998

Self Cite

111

112

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X 2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X 2 subunit was joined to the N terminus of a second P2X 2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X 2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.The extracellular signaling properties of nucleotides are mediated through two distinct families of membrane proteins. These are the P2Y receptors, coupled to G proteins and second messenger pathways, and the P2X receptors, which are ligandgated ion channels (1). Seven subunits of the P2X receptor family (P2X 1-7 ) have been characterized at the molecular level (reviewed in Refs. 2 and 3). These show a broad expression pattern compared with other ligand-gated channels, with the various forms being found in central and peripheral nervous system, different types of immune cells, glands, and smooth and skeletal muscles (4). The P2X receptor subunits can form channels as homomultimers or, in some cases, as heteromultimers (5, 6). The number of subunits in each channel molecule is not known.P2X receptor subunits are 36 -48% identical to one another at the amino acid level. All seven proteins have similar hydrophobicity profiles, with only two hydrophobic regions sufficiently long to span the plasma membrane. These regions display the features often seen in transmembrane segments such as aromatic residues at interfacial regions, and they have an excess of positively charged amino acids at their presumed cytoplasmic ends (7). Together with the absence of signal peptide sequence after the initiating methionine, this suggests that P2X receptors may have intracellular N and C termini, and two transmembrane domains separated by a large extracellular loop (8, 9). This proposed topology differs from that of the nicotinic and glutamate superfamilies of ligand-gated ion channels (reviewed in Ref. 10), but resembles that shown for the pore-forming subunits of epithelial sodium channels (11, 12). There is, however, no detectable similarity of amino acid sequence between P2X re...