Allosteric switching of Ca2+ permeability in the lysosomal ion channel TPC2 underpins biased agonist activation

Abstract

Ion channels possess selectivity filters that are hardwired to ensure the selective passage of ions. Lysosomal two-pore channels are unusual as they are able to switch their cation selectivity in an agonist-specific manner, allowing differential control of organellar activity. TPC2 is permeable to Ca2+ when activated by the calcium-mobilizing messenger NAADP, but largely Na+-selective when activated by the signaling lipid PI(3,5)P2. Co-stimulation increases Ca2+ but not Na+ permeability; however, the molecular basis for these specificity switches is not well understood. Here we show that mutation of TPC2 residues within the distal cytosolic linker, which connects the first voltage-sensing-like domain to the pore, rendered TPC2 largely unable to discriminate its agonists and highly calcium-permeable, even in the presence of PI(3,5)P2. This mutation induced a co-activated-like state by disrupting a network of residues that connects the linker to the activation gate. Such deregulated agonist action increased lysosomal Ca2+ flux and compromised locomotion and viability when expressed in C. elegans. A proximal disease-linked mutation perturbed agonist action in a similar way both in vitro and in vivo. Biased signaling through TPC2 thus proceeds through molecular determinants that are remote from the selectivity filter, affecting Ca2+ permeability, endo-lysosomal integrity and disease. The selectivity of lysosomal TPC2 ion channels for either Ca²⁺ or Na⁺ depends on which agonists activate the channel. This article shows that this plasticity is governed by a distal allosteric network that allows TPC2 to discriminate between agonists and generate specific lysosomal ion fluxes. Ion selectivity of TPC2 can be controlled by residues outside the canonical selectivity filter. Gain-of-function mutations reduce discrimination between NAADP- and PI(3,5)P₂-dependent signalling modes. Mutant channels exhibit enhanced Ca²⁺ permeability and adopt a co-activated-like state. Ion selectivity of TPC2 can be controlled by residues outside the canonical selectivity filter. Gain-of-function mutations reduce discrimination between NAADP- and PI(3,5)P₂-dependent signalling modes. Mutant channels exhibit enhanced Ca²⁺ permeability and adopt a co-activated-like state. A distal allosteric network controls agonist-specific ion selectivity.