Molecular Dynamics Guided Analysis of Ligand Activation, Ion Permeation and Subtype-specific Ligand Interactions in P2X Receptors

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P2X receptors represent a family (P2X1R–P2X7R) of homo- or heterotrimeric ligand-gated ion channels that mediate the depolarizing influx of cations into cells upon binding of extracellular ATP and are interesting targets for new drugs. In the past 3 years, significant advances in our understanding of the molecular function, modulation, and structure of the P2XRs have been made. We identified (i) an ionic-lock mechanism that stabilizes the closed-state conformation of the agonist-binding site of the P2X2R, which is released by ATP binding; (ii) gating conformational changes of the P2X3R; and (iii) subtype-specific interactions between ligands and P2XRs. The recent releases of the first high-resolution X-ray crystal structures of mammalian P2XRs, the human P2X3R and panda P2X7R are crucial for the continuation of the project. The open-, closed-, and desensitized-state hP2X3R structures that for the first time include intracellular domains, provide an unprecedented opportunity to reliably bridge these structures with functional measurements via extensive all-atom molecular dynamics (MD) simulations. In the first grant period, we proved that the combination of structure-guided mutagenesis and electrophysiological analysis is a highly efficient way of detecting and validating molecular interactions. We expect MD stimulations to greatly improve the structure-based guidance of site-directed mutagenesis for electrophysiological experiments. In continuation of the first grant period, main objectives of the second period are to identify the dynamic molecular mechanisms of ligand activation and gating, along with the molecular mechanisms responsible for ion permeation, conductance, and selectivity. A related objective is to identify the molecular determinants of subtype-specific ligand interactions to understand how P2XRs can be selectively targeted by drugs. To this end, we want to comprehensively use MD simulations, ligand-docking, and virtual screening simulations. Hypotheses deduced from these analyses will be validated by site-directed mutagenesis in combination with electrophysiological analysis at the macroscopic and single channel levels. Electrophysiological data will provide iterative feedback for adapting MD simulations with the aim of understanding the functional dynamics of ligand binding-coupled ion flow in P2XRs in atomic detail.