Membrane Pore Formation Unveiled by ∞RETIS Path Sampling: From Thinning to Flip-Flop

Pore formation in lipid bilayers plays a vital role in membrane fusion, transport, and signaling. Yet, its detailed mechanism remains elusive due to the limitations of conventional simulation methods. To overcome this, we apply a newly developed path sampling technique, the asynchronous and infinite swap version of Replica Exchange Transition Interface Sampling (∞RETIS), to study pore formation in a dimyristoylphosphatidylcholine (DMPC) bilayer modeled with the CHARMM36m force field. Our results reveal a sequence of tightly coupled events: pore nucleation sites are determined by early-stage thinning, and the progress into a metastable pore requires a combination of polar defects and close proximity between lipids across opposite leaflets. Using Inf-init, an initiation protocol based on ∞RETIS, rare trajectories can be generated starting directly from equilibrium simulations. Inf-init and ∞RETIS simulations reveal that lipid flip-flop occurs exclusively via local membrane thinning, and pore closure often results in asymmetric lipid distributions.