Mechanistic Insight Into The Activation Of P2X4 Receptors

ATP-gated P2XRs (purinergic receptors) are a class of widely distributed membrane ionotropic receptors found exclusively in eukaryotes, expressed throughout the human body including nervous, cardiovascular and immune systems, and are implicated in a wide range of physiological processes including synaptic transmission, smooth muscle contraction, taste, nociception and inflammation. The resolvation of crystal structure of zebra fish P2X4 receptor in both apo and open states provides important information for further study of this ligand-gated ion channels. In this study, we discussed how ATP-binding induced coordinated movements of a series of structural elements, and finally resulted in the activation of P2X4 receptor. By comprehensive usage of molecular modeling, mutagenesis, and electrophysiological approaches, we proposed a possible activation mechanism of P2X4, named as the’lever’principle. During 300-ns molecular dynamics (MD) simulations of the apo structure of P2X4, a spontaneous closure of the ATP-binding site jaw in the absence of ATP was observed. But this conformational change was not identical to that of the open state, especially the left flipper (LF) and dorsal fin (DF), and did not result in spontaneous opening of the channel. In order to uncover the specific mechanism of the P2X4 activation, we used molecular docking and mutation at resting state of the ATP-binding model, and found that ATP presented a’U’ type structure, interacting with amino acid residues including R298 (β12), K316 (β13), N296 (β12), R298 (β12), K70 (β1), K72 (β1), K193 (β1) and T189 (β8). The hard body domain was just like a’lever’, two ATP contacted with the β1-β8 and β12-β13 of each subunit of this receptor, respectively. The binding of ATP induced downward force exerted upon β12-β13 and upward force applied on β1-β8, both of which acted as the driving force of the ’lever’ motion. Meanwhile, ATP-binding also caused rearrangement of K193 (β8), V291 (LF), L217 (DF) and L208 (LF), and these changes finally lead to channel opening. ATP-binding caused the tightening of the ATP binding site, and coordinated the movement of the LF, DF and head domain. These conformational changes were coupled to the allosteic changes of transmembrane regions via loop structures of post-TM1 and pre-TM2, leading to channel opening. This study provides us a new insight into channel gating of P2X receptor.