The Allostery And Allosteric Regulation Of The Left Flipper Domain During Channel Gating Of P2X Receptors

P2X receptors,as ATP-gated non-selective trimeric ion channels,are permeable to multiple ions,including Na⁺,K⁺and Ca²⁺.Comparing with other ion channel families,P2X receptors are distinct in their unique gating properties and pathophysiological functions,and have attracted vast research attentions as potent and novel drug targets for various disorders such as neuropathic pain,multiple sclerosis,rheumatoid arthritis and thrombus.Several small molecules targeting specific P2X subtypes have entered into main phases of clinical trials.However,many questions regarding to the gating mechanisms of P2X remain unsolved and exploring the gating process of the channels will contribute to developing novel allosteric drugs targeting P2X related disorders.The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains,which marks the beginning of the post-structure era of P2X research at atomic level.Here,we identify coordinated allosteric changes in the left flipper(LF)and dorsal fin(DF)domains that couple ATP-binding to channel gating.Engineered disulphide crosslinking or zinc bridges between the LF and DF domains that constrain their relative motions significantly influence channel gating of P2X4 receptor,confirming the essential role of these allosteric changes.Using multidisciplinary approaches,we proposed that ATP-bound induced conformational changes of the LF domain enable it to establish intersubunit physical couplings among the DF and two lower body domains,which acts as an integrated structural element that is stringently required for the channel gating of P2X4 receptor.Moreover,weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreases or increases the open probability of the receptors,respectively.Furthermore,we identify a negative allosteric site on P2X3 receptors,fostered by the LF and lower body domains.It is essential for the inhibitory effects of several derivatives of AF-353,including AF-219,a drug candidate that is under the phase II clinical trial for refractory chronic cough and idiopathic pulmonary fibrosis.Our studies provide new mechanistic insights into P2X receptors activation and a strategy in the development of new allosteric drugs of P2 X receptors.