P2X purinoreceptors

P2X purinoreceptors

1. Ligand Gated ion channels

   • Monovalent Cation Channel
     • i.e. single + charge
     • some have Ca2+ and anion permeability
   • Channels form as multimers of several subunits
     • Homomeric vs. heteromeric
       • P2X Receptors Flashcards
     • 7 Genes in vertebrates
       • Each ~40-50% identical in amino acid sequence
         • 384-595 aa long
       • Chromosome 12
         • Adjacent to the CDKII genes
       • possess no sequence homology with other proteins
   • Ligand
     • Open in response to binding of extracellular ATP
     • Non-endogenous Agonist
       • aB-methylene ATP

2. Distribution

3. ATP

   • Extracellular ATP acts on cell surface receptors of the P2X receptor
   • rapidly degraded by a series of cell surface enzymes to ADP, AMP, and adenosine
     • Adenosine is then taken back into cells via a specific transporter

4. Structure

   • 2 Transmembrane regions
     • Interconnecting residues make up a substantial extracellular loop
       • 3 glycosylation sites
         • All the P2X receptor subunits have consensus sequences forN-linked glycosylation (Asn-X-Ser/Thr)
         • Receptors in which any two of the three sites are glycosylated appear at the cell surface and are fully functional. Receptors in which only one site is glycosylated give barely detectable currents in response to ATP, and channels with no sites glycosylated give no current.
         • Splice variants alter no. of glyc sites
       • 10 conserved cysteine residues
         • thought that such cysteines in an extracellular location would be oxidized and thus contribute to the tertiary structure of the protein by disulfide bond formation; there is no direct evidence for this in the sense that treatment with reducing agents has no effect on channel function
         • 4 close cys residues interact promiscuously
       • 3 Histidine residues responsible for zinc and proton binding
   • The NH2 and COOH termini are cytoplasmic
     • The COOH-terminal regions diverge in sequence considerably
     • Thr-18 phosphorylated by PKC at intracellular N terminal
     • Ser-431 phosphorylated by PKA (intracellular C termini)

5. Evidence

   • Structure
     • P2X2 receptor currents are potentiated by acidic pH and zinc
       • Histidine-alanine substitutions in Xenopus oocytes
         • 2fold change in EC50, decreasing pH from 7.5 to 6.5
     • The consequences of removal (by tunicamycin) or prevention (by mutagenesis) of glycosylation have been studied
       • Receptors in which any two of the three sites are glycosylated appear at the cell surface and are fully functional
         • detected by immunohistochemistry of a COOH-terminal epitope tag], or immunoprecipitation of cell surface membrane protein labeled with sulfo-NHS-LC-biotin labeling
       • Tunicamycin blocks N-linked glycosylation
   • Multimerisation
     • Functional expression studies
     • cross-linked NH2-terminally tagged P2X1 and P2X3 receptors in intact oocytes
       • trimerization occurred in the endoplasmic reticulum
       • P2X1 identified as a homo-trimer
     • polyacrylamide gel electrophoresis was used to estimate the molecular mass of the P2X1receptor
     • Nickel-affinity chromatografy of purified P2X2
       • photoaffinity labeled with [α-32P]ATP
     • coimmunoprecipitation of epitope-tagged subunits after expression in HEK293 cells

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