Plasticity of the selectivity filter is essential for permeation in lysosomal TPC2 channels.

Two-pore channels are pathophysiologically important Na- and Ca-permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P, they are more Na-selective than when gated by the Ca mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na over Ca. Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions.