Identification And Mechanism Study On The Agonist Of Mechano-gated Piezo1 Channel

Mechanosensation plays critical roles in the living process of biological organism.The mechano-gated ion channels function as key mechanotransducers that convert mechanical stimulation into electrochemical signals rapidly,therefore have important roles from prokaryotic to eukaryotic organism.Piezo family proteins recently have been demonstrated to be the first class of the bona-fide mechano-gated cation channel in mammals.The mouse Piezo1 of 2547 residues forms a three-bladed,propeller-like homo-trimer complex comprising a central ion-conducting pore-module,three propeller-like structures that might serve as mechanotransduction-modules and the connecting beam modules.Piezo1 channel represents a prototype of mammalian mechanosensitive cation channels have been confirmed to be critical for various mechanotransduction processes such as vascular development and cell migration.However,its mechanogating mechanisms remain unclear.Taking advantage of high throughput drug screening,we have identified a novel set of Piezo1 chemical activator,termed Jedi,distinct from the known activator Yoda1.After structure activity relationship study,we determined to focus on Jedi1.Single cell calcium imaging and electrophysiology study show Jedi can evoke piezo1-mediated calcium influx and inward current.Surface plasmon resonance assay reveals Jedi bind to the N-terminal propeller-resembling structure instead of the C-terminal ion-conducting pore region located 100 angstrom away.Combine the structure property of Jedi1 and electrophysiology characterization,we find Jedi1 act on the extracellular side of Piezo1.Deleting two extracellular loop regions 657-677 and 870-921 abolishes Jedi-,but not Yoda1-induced activation of Piezo1.Furthermore,combining Piezo1 structural information and cross-linking and LC-MS/MS anslysis,we mapped the sequence of beam region.Through a series of alanine scanning of beam residues,we identified L1342 and L1345 residues,which mutation abolished both Jedi1 and Yoda1 activation effect.Interestingly,eclectrophysiology data shows the identified two extracellular loops and two residues also decreased the mechanosensitive current of Piezo1,without affect the binding of Jedi1 on Piezo1,indicating long-distance allosteric gating.In conclusion,we identified new chemical activators of Piezo1 and revealed a lever-like mechanotransduction pathway for long-distance chemical-and mechano-gating of the mechanosensitve Piezo1 channel,providing novel tools and insights for further investigating of Piezo channels.