Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.
The elementary release events underlying inositol 1,4,5‐trisphosphate (InsP3)‐mediated calcium signalling were investigated in Xenopus oocytes by means of high‐resolution confocal linescan imaging together with flash photolysis of caged InsP3. Weak photolysis flashes evoked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, spatial spread and amplitude of these elementary events varied widely. Event durations (at half‐maximal amplitude) were distributed exponentially between about 100 and 600 ms. Fluorescence magnitudes (F/F0 of Oregon Green 488 BAPTA‐1) showed a skewed distribution with a peak at about 1.5 and a tail extending as high as 3.5. Individual release sites exhibited both small events (blips) and large events (puffs). The spatiotemporal distribution of calcium signals during puffs was consistent with calcium diffusion from a point source (< a few hundred nanometres), rather than with propagation of a microscopic calcium wave. Estimates of the calcium flux associated with individual events were made by integrating fluorescence profiles along the scan line in three dimensions to derive the ‘signal mass’ at each time point. The smallest resolved events corresponded to liberation of < 2 × 10−20 mol Ca2+, and large events to about 2 × 10−18 mol Ca2+. The rise of signal mass was more prolonged than that of the fluorescence intensity, suggesting that calcium liberation persists even while the fluorescence begins to decline. Rates of rise of signal mass corresponded to Ca2+ currents of 0.4‐2.5 pA. Measurements of signal mass from different events showed a continuous, exponential distribution, arising through variability in magnitude and duration of calcium flux. We conclude that localized calcium transients in the oocyte represent a continuum of events involving widely varying amounts of calcium liberation, rather than falling into separate populations of ‘fundamental’ and ‘elementary’ events (blips and puffs) involving, respectively, single and multiple InsP3 receptor channels. This variability probably arises through stochastic variation in both the number of channels recruited and the duration of channel opening.
Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca2+-dependent trafficking in Parkinson disease.
The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca 2+ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoformspecific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca 2+ release was also impaired using either a Rab bindingdefective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease. Ca 2+ signaling | lysosome | Xenopus T wo-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca 2+ and Na + channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca 2+ /Na + channel with cytosolic NH 2 and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membranespanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2-7) that may support such a lengthy evolutionary pedigree. In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca 2+ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4-6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca 2+ release from "acidic stores." The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na + channels regulated by the endolysosomal phosphoinositide PI(3,5)P 2 . Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactom...
We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activation; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provided by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind before the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate.
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