Bibliographic Details
| Title: |
Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors. |
| Authors: |
Marquez-Klaka, Benjamin1, Rettinger, Jürgen2, Nicke, Annette1 anicke@gwdg.de |
| Source: |
European Biophysics Journal. Mar2009, Vol. 38 Issue 3, p329-338. 10p. 2 Diagrams, 1 Chart, 1 Graph. |
| Subject Terms: |
*CATIONS, *CYSTEINE proteinases, *BINDING sites, *DIMERS, *PROTEINASES |
| Abstract: |
P2X receptors are ATP-gated 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 cross-linking between neighbouring P2X1 subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit 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. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X2K69C and P2X2F289C mutants. Likewise, co-expression of P2X1K68C and P2X2F289C, but not P2X1F291C and P2X2K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X3 or P2X4 mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. 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 function. [ABSTRACT FROM AUTHOR] |
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