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

Abstract

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...

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

“…The N-glycosylation sites within the ectodomain are also essential for trafficking and contribute to the appropriate functionality of P2X1R [37], P2X2R [34,46], P2X4R [23], P2X6R [25], and P2X7R [29]. For P2X1R, any two out of the four naturally occurring N-glycans are sufficient for robust expression of functional receptors at the cell surface [37].…”

“…For P2X1R, any two out of the four naturally occurring N-glycans are sufficient for robust expression of functional receptors at the cell surface [37]. Deletion of all three glycosylated asparagines of the P2X2 subunits induces an intracellular retention and a complete loss of function of the receptors [34,46]. Glycosylation is also important for P2X4R localization on the plasma membrane [23].…”

P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region

“…The extracellular loop contains conserved cysteine, lysine, and glycine residues along with a number of potential N-linked glycosylation sites, all of which contribute to the structural constraints required for ATP binding (11)(12)(13). The C terminus of the P2X7R is 120 amino acids longer than any of the other P2X members.…”

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

“…Different mutations that alter surface expression of P2X subunits have also been reported. Disrupting some of the conserved disulfide bridges of the extracellular loop of P2X 1 subunits alters their normal trafficking to the cell surface (21); similarly, deletion of the three glycosylated asparagines of the P2X 2 subunits induces an intracellular retention and a complete loss of function of the receptors (5,22). However, these mutations are likely to alter the folding of the protein, and the trafficking defects might rather be due a failure to pass the quality check test that normally takes place in the endoplasmic reticulum than due to a trafficking defect per se.…”

“…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).…”