The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca2+ release channels

In this review, we describe the background and implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels gated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Their localisation to the endolysosomal system highlights a new function for these organelles as targets for NAADP-mediated Ca(2+) mobilisation. In addition, we describe how TPCs may also trigger further Ca(2+) release by coupling to the endoplasmic reticular stores through activation of IP(3) receptors and ryanodine receptors

Ca(2+) signals mediated by Ins(1,4,5)P(3)-gated channels in rat ureteric myocytes.

Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors

IMPEACHMENT DA PRESIDENTA DILMA ROUSSEFF: questões de gênero e sentidos propostos à imagem pública, nas capas de Veja, IstoÉ, Época e Carta Capital

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Carbachol triggers RyR-dependent Ca2+ release via activation of IP3 receptors in isolated rat gastric myocytes

Possible interactions between different intracellular Ca2+ release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca2+ signals. Spontaneous, local Ca2+ transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 μm) but not by the inositol 1,4,5-trisphosphate receptor (IP3R) blocker, 2-aminoethoxydiphenyl borate (100 μm), identifying them as Ca2+ sparks. Caffeine (10 mm) and carbachol (10 μm) initiated Ca2+ release at sites which co-localized with each other and with any Ca2+ spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml−1) both inhibited the global cytoplasmic [Ca2+] transient evoked by carbachol, confirming that it was IP3R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca2+ store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP3Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca2+ release through co-operation between IP3Rs and RyRs, and implicate IP3Rs in the regulation of Ca2+ store content

Crosstalk between ryanodine receptors and IP3 receptors as a factor shaping spontaneous Ca2+-release events in rabbit portal vein myocytes

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca2+-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca2+ indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca2+-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca2+ sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F0) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 μm) for the 230 localised Ca2+-release events analysed. The existence of two populations of Ca2+-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca2+-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3–5 min exposure to 1–5 μm nicardipine, but were abolished after 10–15 min exposure to 50–100 μm ryanodine, 0.1 μm thapsigargin or 10 μm cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, 10 μm xestospongin C or 30 μm 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 μm U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca2+-release domains giving rise to spontaneous [Ca2+]i waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP3 receptors to Ca2+, which can give rise to ‘loose coupling’ between release sites. These results suggest that activation of IP3 receptors (due to the tonic activity of PLC and ongoing production of IP3) recruits neighbouring domains of ryanodine receptors, leading to larger Ca2+ releases and saltatory propagation of [Ca2+]i waves in portal vein myocytes