A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

• Published in: The Journal of biological chemistry 2010-05, Vol.285 (21), p.15805-15815
• Main Authors: Jiang, Ruotian, Martz, Adeline, Gonin, Sophie, Taly, Antoine, de Carvalho, Lia Prado, Grutter, Thomas
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Allostery; Amino Acid Substitution; Animals; Biochemistry, Molecular Biology; Cell Line; Cysteine-mediated Cross-linking; Disulfides - metabolism; Gating; Humans; Ion Channel Gating - physiology; Ion Channels; Life Sciences; Ligand-gated Ion Channel; Molecular Biophysics; MTS Compound; Mutation, Missense; Neurotransmitter Receptors; P2X Receptor; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits - genetics; Protein Subunits - metabolism; Purinergic Receptor; Rats; Receptor Structure-Function; Receptors, Purinergic P2 - chemistry; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2X2; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.101980

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu63 and Arg274 produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu63 and Arg274 contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu63 and Arg274, allowing the transmembrane helices to open the channel.