Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones
ABSTRACT The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.
Full-textDOI: · Available from: Alexei Verkhratsky, Jul 29, 2015
- SourceAvailable from: Thomas Taylor-Clark
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- "By inhibiting the SERCA pump with thapsigargin or by activating the ryanodine receptor with caffeine, it is possible to induce the release of Ca 2þ specifically from the ER. In our studies of vagal dissociated neurons, thapsigargin evoked a Ca 2þ transient, consistent with numerous reports of a functional ER within the soma of sensory neurons (Cohen and Moore, 1997; Cordoba-Rodriguez et al., 1999; Gover et al., 2007; Nicolson et al., 2002; Solovyova et al., 2002; Taylor-Clark et al., 2005). Fig. 4 – Representative data of single neuron RT-PCR detection of Orai channels. "
ABSTRACT: Vagal sensory nerves innervate the majority of visceral organs (e.g., heart, lungs, GI tract, etc) and their activation is critical for defensive and regulatory reflexes. Intracellular Ca is a key regulator of neuronal excitability and is largely controlled by the Ca stores of the endoplasmic reticulum. In other cell types store-operated channels (SOC) have been shown to contribute to the homeostatic control of intracellular Ca. Here, using Ca imaging, we have shown that ER depletion in vagal sensory neurons (using thapsigargin or caffeine) in the absence of extracellular Ca evoked Ca influx upon re-introduction of Ca into the extracellular buffer. This store-operated Ca entry (SOCE) was observed in approximately 25-40% of vagal neurons, equally distributed among nociceptive and non-nociceptive sensory subtypes. SOCE was blocked by Gd but not by the Orai channel blocker SKF96365. We found Orai channel mRNA in extracts from whole vagal ganglia, but when using single cell RT-PCR analysis we found only 3 out of 34 neurons expressed Orai channel mRNA, indicating that Orai channel expression in the vagal ganglia was likely derived from non-neuronal cell types. Confocal microscopy of vagal neurons in 3 day cultures demonstrated rich ER tracker fluorescence throughout axonal and neurite structures and ER store depletion (thapsigargin) evoked Ca transients from these structures. However, no SOCE could be detected in the axonal/neurite structures of vagal neurons. We conclude that SOCE occurs in vagal sensory neuronal cell bodies through non-Orai mechanisms but is absent at nerve terminals.Brain research 03/2013; 1503:7-15. DOI:10.1016/j.brainres.2013.02.002 · 2.83 Impact Factor
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- "Xestospongin C also blocked the adverse effect of a presenilin-1 mutation in rendering neurons vulnerable to being damaged by the volatile anesthetic isoflurane (Liang et al., 2008). However, additional actions of xestospongin C on ER Ca 2ϩ handling have been reported, including inhibition of SERCA pumps and depletion of Ca 2ϩ stores without inhibiting IP 3 -induced Ca 2ϩ release in sensory neurons (Solovyova et al., 2002a). Another antagonist at IP 3 R that has been widely used to elucidate the involvement of Ca 2ϩ release from IP 3 sensitive ER stores in experimental models is 2-amino- ethoxydiphenyl borate (2-APB). "
ABSTRACT: The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle capable of integrating multiple extracellular and internal signals and generating adaptive cellular responses. It plays fundamental roles in protein synthesis and folding and in cellular responses to metabolic and proteotoxic stress. In addition, the ER stores and releases Ca(2+) in sophisticated scenarios that regulate a range of processes in excitable cells throughout the body, including muscle contraction and relaxation, endocrine regulation of metabolism, learning and memory, and cell death. One or more Ca(2+) ATPases and two types of ER membrane Ca(2+) channels (inositol trisphosphate and ryanodine receptors) are the major proteins involved in ER Ca(2+) uptake and release, respectively. There are also direct and indirect interactions of ER Ca(2+) stores with plasma membrane and mitochondrial Ca(2+)-regulating systems. Pharmacological agents that selectively modify ER Ca(2+) release or uptake have enabled studies that revealed many different physiological roles for ER Ca(2+) signaling. Several inherited diseases are caused by mutations in ER Ca(2+)-regulating proteins, and perturbed ER Ca(2+) homeostasis is implicated in a range of acquired disorders. Preclinical investigations suggest a therapeutic potential for use of agents that target ER Ca(2+) handling systems of excitable cells in disorders ranging from cardiac arrhythmias and skeletal muscle myopathies to Alzheimer disease.Pharmacological reviews 09/2011; 63(3):700-27. DOI:10.1124/pr.110.003814 · 18.55 Impact Factor
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- "A screen of 2-APB analogues with selectivity for store-operated Ca 2þ entry may yet also provide IP 3 R-selective antagonists (Goto et al. 2010). Xestospongins, isolated from an Australian sponge, are high-affinity membrane-permeant inhibitors of IP 3 -evoked Ca 2þ release that do not affect IP 3 binding (Gafni et al. 1997), but they, too, have side effects (Solovyova et al. 2002). High concentrations of caffeine inhibit IP 3 -evoked Ca 2þ release (Parker and Ivorra 1991) without affecting IP 3 binding (Worley et al. 1987), but caffeine also stimulates RyR, inhibits cyclic nucleotide phosphodiesterases, and interferes with many Ca 2þ indicators. "
ABSTRACT: Inositol 1,4,5-trisphosphate receptors (IP(3)R) and their relatives, ryanodine receptors, are the channels that most often mediate Ca(2+) release from intracellular stores. Their regulation by Ca(2+) allows them also to propagate cytosolic Ca(2+) signals regeneratively. This brief review addresses the structural basis of IP(3)R activation by IP(3) and Ca(2+). IP(3) initiates IP(3)R activation by promoting Ca(2+) binding to a stimulatory Ca(2+)-binding site, the identity of which is unresolved. We suggest that interactions of critical phosphate groups in IP(3) with opposite sides of the clam-like IP(3)-binding core cause it to close and propagate a conformational change toward the pore via the adjacent N-terminal suppressor domain. The pore, assembled from the last pair of transmembrane domains and the intervening pore loop from each of the four IP(3)R subunits, forms a structure in which a luminal selectivity filter and a gate at the cytosolic end of the pore control cation fluxes through the IP(3)R.Cold Spring Harbor perspectives in biology 10/2010; 2(12):a004010. DOI:10.1101/cshperspect.a004010 · 8.23 Impact Factor