Intersubunit disulfide crosslinking in homomeric

Authors: Benjamin MarquezKlaka Jürgen Rettinger Annette Nicke

Publish Date: 2008/04/22

Volume: 38, Issue: 3, Pages: 329-338

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Abstract

P2X receptors are ATPgated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits Each subunit contains two transmembrane domains connected by a large extracellular loop We have previously shown that replacement of two conserved residues K68 and F291 by cysteine residues leads to disulfide crosslinking between neighbouring P2X1 subunits Since mutation of these residues results in a reduced ATP potency and cysteine crosslinking is prevented in the presence of ATP we suggested an intersubunit ATP binding site To investigate whether the proximity of these residues is preserved in other P2X subtypes we tested for spontaneous cystine formation between the corresponding P2X2 K69C F289C P2X3 K63C F280C and P2X4 K67C F294C mutants upon pairwise expression in Xenopus laevis oocytes Nonreducing SDSPAGE analysis of the purified receptors revealed a specific dimer formation between P2X2K69C and P2X2F289C mutants Likewise coexpression of P2X1K68C and P2X2F289C but not P2X1F291C and P2X2K69C mutants resulted in dimer formation between the respective subunits Crosslinked P2X1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT Crosslinking was less efficient between P2X3 or P2X4 mutants but could be enhanced by the short cysteinereactive crosslinker MTS2MTS These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X1 are preserved in P2X2 receptors and at one of two possible interfaces in heteromeric P2X1/2 receptors but appears to be redundant for P2X3 and P2X4 receptor functionExtracellular ATP is an autocrine and paracrine messenger molecule as well as a neurotransmitter P2X receptors P2XRs are ATPactivated nonselective cation channels with a high Ca2+ permeability Besides cysloop receptors comprising nicotinic acetylcholine receptors nAChRs serotonine receptor subtype 3 glycine receptors and GABAA and GABAC receptors and ionotropic glutamate receptors iGluRs comprising AMPA kainate and NMDA receptors P2XRs represent a third major family of neurotransmittergated ion channels For both the tetrameric iGluR and the pentameric nAChR families the molecular structure of their agonist binding sites has been obtained by the Xray structural analysis of soluble proteins with high homology to the respective agonist binding sites

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