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.

Background: Biochemical characterization of voltage-dependent anion channel 2 (VDAC2) is limited due to an inability to obtain functional protein. Results:The crystal structure of VDAC2 suggests a dimer interface that is confirmed by double electron-electron resonance and cross-linking. Conclusion: zfVDAC2 has a fractional dimeric population. Significance: VDAC isoforms are structurally similar, but this study has identified a number of hot spots that require further exploration.

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 depolarizationinduced but not caffeine-induced Ca 2؉ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the ␣1S subunit is normal, whereas the ␤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 ␤1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a ␤1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the ␣1S subunit in the absence of ␤1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an Ϸ2-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 ␤1a subunit does not prevent triad targeting of the DHPR ␣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 excitationcontraction coupling in ␤1-null model organisms.calcium channels ͉ excitation-contraction coupling ͉ tetrads ͉ zebrafish E xcitation-contraction (EC) coupling is understood as the signal transduction process connecting membrane depolarization to the contraction of muscle cells. This process is initiated by the concerted action of two Ca 2ϩ channels, the plasmalemmal voltage-gated dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptor (RyR). In junctions of the SR with the plasma membrane (peripheral couplings) or with the transverse tubules (triads), membrane depolarization is sensed by the DHPR, which then triggers RyR opening and Ca 2ϩ release from the SR. In skeletal muscle cells, this signaltransduction is independent of Ca 2ϩ influx through the DHPR (1) but depends on protein-protein interaction between the DHPR and the RyR1 (2, 3). This physical coupling requires the coordinated arrangement of DHPRs and RyR1s in the junctions. In skeletal muscle triads and peripheral couplings, groups of four DHPRs (tetrads) are arranged in orthogonal arrays matching the opposing RyR1 arrays (4). Formation of DHPR tetrads requires the presence of RyR1.The skeletal muscle DHPR complex is composed of the voltage-sensing and pore-forming ␣ 1S subunit and the auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ (5). Targeted deletions of the ␣ 2 ␦-1 and ␥ subunits do not critically interfere with EC coupling function (6, 7). In contrast, ␣ 1S and ␤ 1a subunit null-mutant mice display a lack of EC coupling and, thus, lethal muscle paralysis (8, 9). Although failure o...

Summary Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl−/− embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl−/− hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Sclfl/fl Rosa26Cre-ERT2 embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl−/− endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.