A Piezo1 Open State Reveals a Multi-fenestrated Ion Permeation Pathway

Abstract

19Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation 20 of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as 21 phosphatidylinositol-4,5-bisphosphate (PIP2). Here, to study how Piezo1 opens, we performed 22 tension) POPC membrane (11). We also showed that the dome rapidly flattens when membrane 60 tension is gradually increased. Despite flattening of the arms, however, the pore did not open. 61Since the Piezo1 arms are anticipated to act as mechanical levers, the shorter arms in our 62 truncated model may reduce the output force (on the pore) to the input effort (arm motion). 63Another possibility for the absence of opening motions in the pore may have come from the 64 symmetric property of our simulated bilayer: indeed, electrophysiological recordings showed 65 Piezo1 remains fully closed when reconstituted in symmetrical bilayers but spontaneously opens 66 in asymmetric bilayers containing dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) or 67 lysophosphatidic acid (LPA) in the inner leaflet (12, 13). DOPA and LPA differ in the number of 68 fatty acid tails but are both negatively-charged. Interestingly, the presence of negatively-charged 69 PIP2 or phosphatidylserine (PS) lipids in the inner leaflet also promote channel activation (14-16). 70We thus reasoned that adding the missing arm regions and adding negatively charged PIP2 lipids 71 in the inner leaflet will allow us to computationally capture a Piezo1 open state. 72 73 74RESULTS 75 Piezo1 clustering induces flattening of the Piezo arms 76Our previous proof-of-concept AA simulation showed that the membrane curvature, or lipid dome, 77 imposed by the resting conformation of a truncated Piezo1 takes place over 3 μs (11). To reduce 78 computational time, here we first used a 12 μs Coarse-Grained (CG) Martini simulation to enable 79 rapid lipid diffusion and dome formation while the Piezo1 backbone was kept rigid. We performed 80 these CG simulations on both a symmetrical POPC membrane (PC:PC) and an asymmetrical 81 POPC membrane containing 5% PIP2 in the inner leaflet (PC:PC/PIP2) (Figure 1a, systems I and 82 II). The CG systems were then mapped back to an AA system and simulated it for an additional 83 2 μs (Figure 1a, systems III and IV). In all MD simulations with explicit solvent, the periodic 84 boundary conditions (PBC) create an infinite lattice where the simulated system is infinitely 85

Piezo1 Forms Specific, Functionally Important Interactions with Phosphoinositides and Cholesterol

“…Negative lipids such as PIPs, PIs, PAs, and PSs share common binding sites, as seen in Fig. 4 (more detail is available in Table S2), characterized by the presence of basic residues on the protein surface, some of which are identified in a recent preprint (67). PIPs, having the largest negative charge, tend to have the highest occupancy in many of these sites.…”