Analysis of Structural Rearrangements during P2X1 Receptor Activation by Voltage Clamp Fluorometry

Abstract

P2X receptors (P2XRs) are non-selective cation channels which are activated upon binding of extracellular ATP. They are assembled as homo- or heteromers from three subunits with two transmembrane domains each and show no sequence homology to any other known ion channel or ATP-binding protein. Most recently, the first crystal structure of a P2X receptor has been resolved confirming many predictions that were based on the interpretation of mutagenesis studies. However, the conformational changes governing channel opening, desensitization and recovery remain unknown, as does the exact mode of ATP binding. The P2X1 receptor subtype is characterized by nanomolar affinity for ATP and a rapid desensitization, followed by a prolonged recovery period before reactivation is possible. Here we used voltage clamp fluorometry to identify domains undergoing conformational changes during ligand binding, activation, desensitization and recovery from desensitization of the P2X1 receptor. We have identified six residues in the extracellular domain of the P2X1 receptor that, upon substitution with cysteine, are accessible for TMRM labeling. Upon activation by ATP, five of these mutants showed significant changes in fluorescence. The kinetics of the fluorescence changes could be correlated with receptor activation or desensitization indicating that the changes were reporting discrete conformational changes. In addition, binding of the competitive antagonist NF449 produced a fluorescence change in three of these mutants. In conclusion, our observations provide insight into the conformational changes occurring during the P2X opening and desensitization and suggest that movements in a cysteine rich domain that projects over the supposed ATP binding site are involved in these processes.