Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Neurosci
2007 Feb 07;276:1456-66. doi: 10.1523/JNEUROSCI.3105-06.2007.
Show Gene links
Show Anatomy links
Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor.
Marquez-Klaka B
,
Rettinger J
,
Bhargava Y
,
Eisele T
,
Nicke A
.
???displayArticle.abstract???
P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, two-electrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X(2-1) chimera showed only small ATP-activated currents that, however, increased approximately 60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors.
Barrera,
Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize.
2005, Pubmed
Barrera,
Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize.
2005,
Pubmed
Bean,
ATP-activated channels in rat and bullfrog sensory neurons: current-voltage relation and single-channel behavior.
1990,
Pubmed
Brejc,
Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.
2001,
Pubmed
Clyne,
Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor.
2002,
Pubmed
,
Xenbase
Colquhoun,
Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors.
1998,
Pubmed
Ding,
Single channel properties of P2X2 purinoceptors.
1999,
Pubmed
,
Xenbase
Ennion,
The role of positively charged amino acids in ATP recognition by human P2X1 receptors.
2000,
Pubmed
Ennion,
Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface.
2002,
Pubmed
,
Xenbase
Finger,
ATP signaling is crucial for communication from taste buds to gustatory nerves.
2005,
Pubmed
Freist,
ATP binding site of P2X channel proteins: structural similarities with class II aminoacyl-tRNA synthetases.
1998,
Pubmed
Jiang,
Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor.
2000,
Pubmed
Jiang,
Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor.
2001,
Pubmed
Jiang,
Subunit arrangement in P2X receptors.
2003,
Pubmed
Mahaut-Smith,
ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets.
2000,
Pubmed
,
Xenbase
Nagaya,
An intersubunit zinc binding site in rat P2X2 receptors.
2005,
Pubmed
,
Xenbase
Nakazawa,
Amino acid substitutions from an indispensable disulfide bond affect P2X2 receptor activation.
2004,
Pubmed
,
Xenbase
Nicke,
P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.
1998,
Pubmed
,
Xenbase
North,
Molecular physiology of P2X receptors.
2002,
Pubmed
O'Hara,
The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins.
1993,
Pubmed
Rettinger,
Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations.
2003,
Pubmed
,
Xenbase
Rettinger,
Desensitization masks nanomolar potency of ATP for the P2X1 receptor.
2004,
Pubmed
,
Xenbase
Roberts,
ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists.
2004,
Pubmed
,
Xenbase
Roberts,
Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP.
2006,
Pubmed
,
Xenbase
Roberts,
Molecular properties of P2X receptors.
2006,
Pubmed
Schägger,
Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.
1994,
Pubmed
Werner,
Domains of P2X receptors involved in desensitization.
1996,
Pubmed
,
Xenbase
Wilkinson,
Role of ectodomain lysines in the subunits of the heteromeric P2X2/3 receptor.
2006,
Pubmed
Yan,
Molecular determinants of the agonist binding domain of a P2X receptor channel.
2005,
Pubmed
Yan,
Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate.
2006,
Pubmed
Zemkova,
Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.
2004,
Pubmed
