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

Abstract

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...

Gated Access to the Pore of a P2X Receptor

“…This is because past work shows that the N and C termini are vital for the function of P2X2 receptors (10). For example, deletion of the N terminus completely abolishes functional responses (55), and the C terminus of the P2X2 receptors contains a membrane stabilization motif, the absence of which results in negligible ATP-evoked responses (56). Thus, truncated P2X2 receptors would be of little use for the present electrophysiological experiments as they would not be functional.…”

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

“…The conserved YXXXK motif found in Cterminal of P2XRs other than P2X7R is necessary for their expression at the cell surface [24]. In human P2X7R, a motif located in the distal region of its C-terminus tail is necessary for proper receptor trafficking; progressive deletions of this tail between residues 551 and 581 are nonfunctional and are not found in the plasma membrane [10,41].…”

“…Membrane stability of P2XR channels might be promoted by interactions with intracellular proteins that regulate membrane turnover and degradation of the receptor [18,24]. Another possible mechanism by which the SS mutants could block P2X7R trafficking is a generation of SS bonds between single cysteines and intracellular proteins within the cytoplasm.…”

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane

“…HA tag was inserted at a similar position in an N-terminally flag tagged human TRPV2 channel (between residues A 419 and A 420 ). Amino acid substitutions were generated by using oligonucleotide-directed mutagenesis as previously described [15]. All constructs were verified by DNA sequencing.…”