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The Journal of Neuroscience, February 7, 2007, 27(6):1456-1466; doi:10.1523/JNEUROSCI.3105-06.2007

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Cellular/Molecular
Identification of an Intersubunit Cross-Link between Substituted Cysteine Residues Located in the Putative ATP Binding Site of the P2X₁ Receptor

Benjamin Marquez-Klaka,¹ Jürgen Rettinger,² Yogesh Bhargava,² Thomas Eisele,³ and Annette Nicke¹

¹Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60528 Frankfurt, Germany, ²Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt, Germany, and ³Department of Molecular Pharmacology, RWTH Aachen University, D-52074 Aachen, Germany

Correspondence should be addressed to Dr. Annette Nicke, Max-Planck-Institute for Brain Research, Department of Neurochemistry, Deutschordenstrasse 46, D-60528 Frankfurt, Germany. Email: anicke{at}gwdg.de

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 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.

Key words: ATP binding site; P2X receptor; disulfide cross-linking; subunit interface; cysteine substitution; two-electrode voltage-clamp

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Received July 21, 2006; revised Dec. 5, 2006; accepted Dec. 31, 2006.

Correspondence should be addressed to Dr. Annette Nicke, Max-Planck-Institute for Brain Research, Department of Neurochemistry, Deutschordenstrasse 46, D-60528 Frankfurt, Germany. Email: anicke{at}gwdg.de

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