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      Acidic Amino Acids Impart Enhanced Ca 2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

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          Abstract

          P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca 2+ permeabilities that are amongst the highest of all ligand-gated channels ( Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca 2+ permeability of the P2X 2 receptor ( Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca 2+ currents ( Pf%) of the rat and human P2X 1 and P2X 4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X 1 receptor but had no effect on the lower Pf% of the P2X 2 receptor, suggesting that ionized side chains regulate the Ca 2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X 1 and P2X 4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X 2 receptor. Taken together, the data suggest that COO side chains provide an electrostatic force that interacts with Ca 2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X 1 and P2X 4 receptors is conserved in the P2X 3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His 45 increased Pf% of the P2X 3 channel, suggesting that this positive charge masks the facilitation of Ca 2+ flux by the neighboring Glu 46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca 2+ permeability and flux of some P2X receptors.

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          Most cited references42

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          P2X receptors as cell-surface ATP sensors in health and disease.

          P2X receptors are membrane ion channels activated by the binding of extracellular adenosine triphosphate (ATP). For years their functional significance was consigned to distant regions of the autonomic nervous system, but recent work indicates several further key roles, such as afferent signalling, chronic pain, and in autocrine loops of endothelial and epithelial cells. P2X receptors have a molecular architecture distinct from other ion channel protein families, and have several unique functional properties.
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            Vesicular release of ATP at central synapses.

            Adenosine triphosphate (ATP) acts as a fast excitatory transmitter in several regions of the central nervous system (CNS) including the medial habenula, dorsal horn, locus coeruleus, hippocampus, and somatosensory cortex. Postsynaptic actions of ATP are mediated through an extended family of P2X receptors, widely expressed throughout the CNS. ATP is released via several pathways, including exocytosis from presynaptic terminals and diffusion through large transmembrane pores (e.g., hemichannels, P2X(7) receptors, or volume-sensitive chloride channels) expressed in astroglial membranes. In presynaptic terminals, ATP is accumulated and stored in the synaptic vesicles. In different presynaptic terminals, these vesicles may contain ATP only or ATP and another neurotransmitter [e.g., gamma-amino-butyric acid (GABA) or glutamate]; in the latter case, two transmitters can be coreleased. Here, we discuss the mechanisms of vesicular release of ATP in the CNS and present our own data, which indicate that in central neuronal terminals, ATP is primarily stored and released from distinct pool of vesicles; the release of ATP is not synchronized either with GABA or with glutamate.
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              P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

              P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. blue native PAGE/cross-linking/P2X receptor/quaternary structure.
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                Author and article information

                Journal
                J Gen Physiol
                The Journal of General Physiology
                The Rockefeller University Press
                0022-1295
                1540-7748
                March 2007
                : 129
                : 3
                : 245-256
                Affiliations
                Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO 63104
                Author notes

                Correspondence to Terrance Egan: egantm@ 123456slu.edu

                Article
                200609677
                10.1085/jgp.200609677
                2151611
                17325195
                b1720b97-5f7f-4307-806e-f0837f642d04
                Copyright © 2007, The Rockefeller University Press
                History
                : 9 October 2006
                : 6 February 2007
                Categories
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                Article

                Anatomy & Physiology
                Anatomy & Physiology

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