Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca²⁺-signals and non-selective cation currents, the other weaker Ca²⁺-signals and Na⁺-selective currents. These properties were mirrored by the Ca²⁺-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P₂, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Å resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.

Homozygous zebrafish of the mutant relaxed ( red ^ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca ²⁺ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the α _1S subunit is normal, whereas the β _1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the β _1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a β _1a -null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the α _1S subunit in the absence of β _1a . Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an ≈2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the β _1a subunit does not prevent triad targeting of the DHPR α _1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in β ₁ -null model organisms.

Tightly regulated Ca²⁺ homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca²⁺ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca²⁺ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca²⁺ uptake and accelerates the transfer of Ca²⁺ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca²⁺ sparks and thereby inhibits Ca²⁺ overload-induced erratic Ca²⁺ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca²⁺ uptake in the regulation of cardiac rhythmicity.