Explore the vast range of titles available.

Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P 2 . Whereas NAADP rendered the channel Ca 2+ -permeable and PI(3,5)P 2 rendered the channel Na + -selective, a combination of the two increased Ca 2+ but not Na + flux. Mechanistically, this was due to an increase in Ca 2+ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P 2 mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca 2+ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca 2+ signaling. TPC2 is a lysosomal ion channel permeable to both calcium and sodium ions. Here, the authors show that TPC2 can selectively increase its calcium permeability when simultaneously challenged by both its natural activators- NAADP and PI(3,5)P 2 .

THE increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that follows electrical activity in excitable cells influences various cellular functions. But the measured increase in average cytosolic [Ca 2+ ] i (typically 10 nM per action potential 1–3 ) is often less than the micromolar [Ca 2+ ] i required to activate proteins controlling cell function 4–5 . We now report that clustering of L-type Ca 2+ channels causes [Ca 2+ ] i hotspots of average diameter 7 μm at the neuronal growth cone. At the hotspot, [Ca 2+ ] i changes of the order of 1 μM were recorded during 1-s voltage-clamp depolarizations, whereas a single action potential raised [Ca 2+ ] i by 89 nM. Depolarization will therefore activate enzymes with a micromolar requirement for Ca 2+ at the hotspot. Local morphological changes near the site of the hotspot were induced by action potentials. We observed hotspots in all regions of the growth cone, usually at the base of processes extending from the growth-cone palm, but never in the neurite. The role of voltage-dependent Ca 2+ influx in controlling nerve cell outgrowth has been a puzzle: although raised [Ca 2+ ] i triggers outgrowth of the growth cone margin, neurite elongation requires low [Ca 2+ ] i (refs 6–8). Our results resolve this paradox: electrical activity can selectively raise [Ca 2+ ] i in the growth cone, leaving neurite [Ca 2+ ] i low. Gross [Ca 2+ ] i gradients and localized hotspots have been previously reported in depolarized neurons 3,9–12 . Patch-clamp, toxin-binding and freeze-fracture studies have demonstrated that calcium channels are grouped in clusters 13–17 . However, this is the first report that calcium channel clusters can cause [Ca 2+ ] i hotspots, and that channel clustering has a physiological role.

EXTRACELLULAR events regulate functions in the cell nucleus by means of calcium ions acting through effector enzymes 1–5 . Recently, the traditional view of the nuclear pore as freely permeable to small ions 6,7 has been questioned as a result of reports that nuclear calcium can be regulated independently of cytosolic calcium 8–12 . We have used confocal microscopy of fluorescent Ca 2+ indicators to investigate the Ca 2+ dynamics between cytosol and nucleus in neurons. We find that a previously reported amplification of Ca 2+ changes in the nucleus 13–16 is a measurement artefact. Small changes of cytosolic Ca 2+ cause equally rapid changes in nuclear Ca 2+ , consistent with the free diffusion of Ca 2+ through nuclear pores. In contrast, large cytosolic Ca 2+ increases (above 300 nM) are attenuated in the nucleus. Our results show the nuclear envelope shapes but does not block the passage of Ca 2+ signals from cytosol to nucleus.

We have used fluorescently labelled calmodulins to probe the activity of calmodulin in living dorsal root ganglion cells. Calmodulin labelled with the fluorophore 5-([4,6 dichlorotriazin-2yl]amino)-fluorescein (FL-CaM) does not change its fluorescence when it binds calcium, while calmodulin labelled at lysine 75 with 2-chloro-(6-(4-N,N-diethylamino-phenyl)-1,4,5-triazin-4-yl (TA-CaM), an environment-sensitive probe, increases its fluorescence when it binds calcium. We micro-injected FL-CaM or TA-CaM into rat dorsal root ganglion cells and found that both probes localise to the cell nucleus. In contrast, endogenous cellular calmodulin, in dorsal root ganglion cells as in hippocampal neurones, is predominantly cytosolic unless the neurones are depolarised, then it moves to the nucleus. FL-CaM and TA-CaM, introduced into dorsal root ganglion cells via a patch pipette, also immediately move to the nucleus, indicating that the nuclear localisation is a property of the labelled calmodulins. Although the subcellular distribution of FL-CaM and TA-CaM does not necessarily match that of endogenous calmodulin, we show that FL-CaM can be used as a control for TA-CaM when studying calmodulin activation in different cellular compartments.

We have developed a non-invasive technique to measure intracellular calcium ([Ca2+]i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66+/-22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca2+]i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca2+]i oscillations which may have a functional significance during neuronal development.