Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor.
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.
Marquez-Klaka
Benjamin
B
Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60528 Frankfurt, Germany.
Rettinger
Jürgen
J
Bhargava
Yogesh
Y
Eisele
Thomas
T
Nicke
Annette
A
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000502:physiology*; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006031:Glycosylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058469:Receptors, Purinergic P2X; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014982:Xenopus laevis
D021122:Protein Subunits; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D011993:Recombinant Fusion Proteins; D003553:Cystine; D000255:Adenosine Triphosphate; D003545:Cysteine; D004229:Dithiothreitol