Amino Acid Residues Involved in Gating Identified in the First Membrane-spanning Domain of the Rat P2X2 Receptor

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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...

supporting

confidence: 71%

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

Chaumont

Jiang

Penna

et al. 2004

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“…In contrast to the experiences with TM2, SCAM experiments aimed at elucidating a role for TM1 were less clear, leading to the conclusion that this domain affected channel gating (21,22,72). Later, the region responsible for calcium ion selection was identified in TM2 using mutagenesis coupled with relative ion permeability and fractional calcium current measurements (27,28).…”

Section: Discussionmentioning

confidence: 99%

Gated Access to the Pore of a P2X Receptor

Kracun

Chaptal

Abramson

et al. 2010

P2X receptors (P2X1-P2X7) are cell surface cation channels that transition from closed to open states upon binding ATP. They represent a physiologically and medically important (1), as well as a structurally distinct family of ligand-gated ion channels that are quite different to Cys-loop and ionotropic glutamate receptors (2-5). P2X receptors are found in many species from several phyla, implying important roles in diverse life forms (6, 7). A key goal is to understand how P2X receptors operate at the chemical level.The first P2X receptor genes were cloned in 1994 (8, 9) leading to important progress over the last decade in understanding how P2X receptors function (10, 11) using biophysical and biochemical approaches. P2X subunits are thus known to possess intracellular N and C termini and two transmembrane (TM) 2 segments separated by a large extracellular loop (12-14). The trimeric architecture of P2X receptors is also well established based on subunit concatemers, blue-native PAGE, and atomic force microscopy experiments (15-18). It is also well established that the P2X pore is lined by TM2 (19 -22), with TM1 making little contribution to ion flow (23, 24). Moreover, careful studies of permeation and selectivity have suggested that the same area of TM2 is responsible for both ion selection and channel opening (25-31). Mutational approaches have shed light on the extracellular domain of P2X receptors, including how the ATP binding pocket may form (32), how the 10 conserved cysteines contribute to the fold of the protein (33, 34), and how cations such as Zn 2ϩ profoundly affect receptor function (35)(36)(37)(38). Finally, meticulous analysis of voltage-jump relaxations for ATP-evoked currents combined with mutagenesis has suggested that TM2 may display hinge-like flexibility (39, 40), and recently the P2X4 receptor open pore has been imaged with fast scanning atomic force microscopy (41). Most of these past studies were conducted on rat P2X2 (rP2X2) receptors and have provided a basis to explore P2X receptor pores and the gating process.In 2009, the field was immeasurably advanced by the landmark achievement and report of a crystal structure of the zebrafish (zf) P2X4.1 receptor in the closed state (42). The P2X receptor topology was similar to that of acid-sensing ion channels (42, 43), a possibility that had been raised by earlier modeling studies (44). However, it is important to note that the extracellular domains of P2X and acid-sensing ion channels are distinct with little sequence or structural homology, whereas the pore domains share similar architectures (43). The availability of direct structural information for P2X receptors substantiated most of the aforementioned mutational studies and for the first time provided a framework to understand their atomic origins (5). Of most relevance here, it directly revealed that the P2X pore was indeed formed by TM2 ␣-helices arranged around a 3-fold molecular axis of symmetry. These were arranged ϳ45°to the membrane normal and crossed about halfway along their l...

“…Murine P2X7aRs and P2X7kRs have identical pore forming TM2s but divergent Pf %, and differ only in the primary sequences of their N termini and TM1s. Site-directed mutagenesis of the TM1 of the rat P2X2R affects gating (96,97) to a greater extent than the Pf % (62), suggesting that TM1 plays little or no role in modulation of the Ca 2ϩ component of the ATP-gated current. We assume that this condition holds true for P2X7Rs too, and we plan to test this hypothesis in future experiments.…”

Section: Discussionmentioning

confidence: 99%

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Liang¹,

Samways²,

Wolf³

et al. 2015

Background: Ca 2ϩ triggers many of the actions of extracellular ATP. Results: We measured the Ca 2ϩ component of the ATP-gated non-selective cation current of P2X7 receptors. Conclusion: Ca 2ϩ flux varied with the species of origin, the splice variant, and the agonist concentration. Significance: Our results suggest that domains that lie outside of the pore regulate the Ca 2ϩ flux of P2X7 receptors.