共查询到20条相似文献,搜索用时 515 毫秒
1.
《European journal of cell biology》2019,98(2-4):116-123
Lysosomal Ca2+ release channel TRPML1 has been suggested to regulate lysosome size by activating calmodulin (CaM). To further understand how TRPML1 and CaM regulate lysosome size, in this study, we report that inhibiting mTORC1 causes enlarged lysosomes, and the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. We also show that lysosome vacuolation induced by inhibiting TRPML1 is corrected by mTORC1 upregulation, and the facilitating effect of TRPML1 on the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. In the meantime, lysosome vacuolation induced by inhibiting CaM is corrected by mTORC1 upregulation, and mTORC1 overexpression corrects the inhibitory effect of CaM antagonist on the recovery of enlarged lysosomes. Conversely, the vacuolation induced by suppressing mTORC1 is not corrected by upregulating CaM. These data suggest that mTORC1 functions downstream of TRPML1 and CaM to regulate lysosome size. Together with our recent finding showing that TRPML1, CaM and mTORC1 form a macromolecular complex to control mTORC1 activity, we suggest that TRPML1 and CaM control lysosome fission through regulating mTORC1, identifying an mTORC1-dependent molecular mechanism for lysosomal membrane fission. 相似文献
2.
Abdelilah Arredouani Margarida Ruas Stephan C. Collins Raman Parkesh Frederick Clough Toby Pillinger George Coltart Katja Rietdorf Andrew Royle Paul Johnson Matthias Braun Quan Zhang William Sones Kenju Shimomura Anthony J. Morgan Alexander M. Lewis Kai-Ting Chuang Ruth Tunn Joaquin Gadea Lydia Teboul Paula M. Heister Patricia W. Tynan Elisa A. Bellomo Guy A. Rutter Patrik Rorsman Grant C. Churchill John Parrington Antony Galione 《The Journal of biological chemistry》2015,290(35):21376-21392
Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells. 相似文献
3.
Kalina Dimova Noa Lipstein Donghan Lee Stefan Becker Nils Brose Olaf Jahn Christian Griesinger 《The EMBO journal》2010,29(3):680-691
Ca2+ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca2+–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca2+ signals. We solved the structure of Ca2+4–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca2+2–CaM complex. The Ca2+ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca2+–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca2+]i, whereas the N‐module acts as a sensor at micromolar [Ca2+]i. This Ca2+/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca2+‐dependent modulation of short‐term synaptic plasticity. 相似文献
4.
Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores 总被引:1,自引:0,他引:1
Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca2+ by pretreatment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells. 相似文献
5.
Dong Wang Daniel Epstein Ossama Khalaf Sankaranarayanan Srinivasan W. Ryan Williamson Amir Fayyazuddin Florante A. Quiocho P. Robin Hiesinger 《The Journal of cell biology》2014,205(1):21-31
Most chemical neurotransmission occurs through Ca2+-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca2+ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca2+ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca2+ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H+-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca2+–Calmodulin (CaM)-dependent manner. Ca2+–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca2+-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca2+–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release. 相似文献
6.
Bao-Guang Hua Richard W. Mercier Raymond E. Zielinski Gerald A. Berkowitz 《Plant Physiology and Biochemistry》2003,41(11-12):945-954
A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca2+–dependent manner with a Kd of 7.6 nM and a Ca2+ binding Kd of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca2+. Increasing cytosolic Ca2+ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca2+/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca2+ can act in an integrated fashion to gate currents through these plant ion channels. 相似文献
7.
Zhiping P. Pang Wei Xu Peng Cao Thomas C. Südhof 《The Journal of biological chemistry》2010,285(44):33930-33939
Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca2+ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca2+ binding to an unidentified secondary Ca2+ sensor. To test whether CaM could be the enigmatic Ca2+ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca2+ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca2+/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis. 相似文献
8.
Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca2+ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca2+ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca2+ release channel (RyR1). Here we report that SPC also displaces Ca2+-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca2+CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [3H]ryanodine binding assays. In the presence of Ca2+CaM, however, the addition of SPC did not reduce [3H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca2+CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca2+ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely. 相似文献
9.
Justine Yu Elena Deliu Xue-Quian Zhang Nicholas E. Hoffman Rhonda L. Carter Laurel A. Grisanti G. Cristina Brailoiu Muniswamy Madesh Joseph Y. Cheung Thomas Force Mary E. Abood Walter J. Koch Douglas G. Tilley Eugen Brailoiu 《The Journal of biological chemistry》2013,288(31):22481-22492
The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to
Ca2+ signaling. Low levels of GPR55 expression in the heart have been reported.
Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed
both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we
used calcium and voltage imaging and extracellular administration or intracellular microinjection of
GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI
triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation
at the sarcolemma elicits, on one hand, Ca2+ entry via L-type
Ca2+ channels and, on the other, inositol 1,4,5-trisphosphate-dependent
Ca2+ release. The latter signal is further amplified by
Ca2+-induced Ca2+ release via ryanodine receptors. Conversely,
activation of GPR55 at the membrane of intracellular organelles promotes Ca2+
release from acidic-like Ca2+ stores via the endolysosomal NAADP-sensitive
two-pore channels. This response is similarly enhanced by Ca2+-induced
Ca2+ release via ryanodine receptors. Extracellularly applied LPI produces
Ca2+-independent membrane depolarization, whereas the Ca2+
signal induced by intracellular microinjection of LPI converges to hyperpolarization of the
sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor
regulating cardiac function at two cellular sites. This work may serve as a platform for future
studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders. 相似文献
10.
Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca2+ and Na+ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca2+ release channels that respond to activation by the Ca2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled. 相似文献
11.
Xu Wang Liang Wen Xiong Amina El Ayadi Darren Boehning John A. Putkey 《The Journal of biological chemistry》2013,288(3):2040-2048
PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca2+ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca2+ binding activity, which may modulate Ca2+ binding to CaM by stabilizing an initial Ca2+-CaM complex or by electrostatically steering Ca2+ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca2+ dynamics, we tested the hypothesis that it influences ligand-dependent Ca2+ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca2+ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca2+ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca2+ binding to CaM greatly inhibit its effect on ATP-induced Ca2+ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca2+ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling. 相似文献
12.
Peter Svenssen Munksgaard Thomas Vorup‐Jensen Jesper Reinholdt Carl Martin Söderström Knud Poulsen Jens Leipziger Helle A. Praetorius Marianne Skals 《Cellular microbiology》2012,14(12):1904-1920
Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat‐in‐toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α‐haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA‐mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA‐mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS‐exposure is preceded by increments in the intracellular Ca2+ concentration ([Ca2+]i). Interestingly, LtxA‐mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA‐induced [Ca2+]i increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2‐mediated auto‐ and paracrine signalling may prevent LtxA‐mediated cell damage. 相似文献
13.
Chr Zrb Katja D. Brunner M. Perbandt Chr Betzel G. Wagner 《Plant biology (Stuttgart, Germany)》1998,111(5):346-353
The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca2+-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca2+ buffers in the PDE test, affinity to Ca2+ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (Kact) in the PDE test for Mougeotia CaM relative to maize CaM, while the Kact of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling. 相似文献
14.
《Journal of molecular biology》2019,431(7):1440-1459
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts. 相似文献
15.
The 5′-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca2+ entry (SOCE). However, whether mAChRs may control AMPK activity by regulating additional mechanisms beyond SOCE remains to be investigated. In the present study we examined the effects of mAChRs on AMPK when SOCE was induced by the sarco–endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. We found that in SH-SY5Y cells depleted of Ca2+ by thapsigargin, the re-addition Ca2+ to the medium stimulated AMPK phosphorylation at Thr172, which is required for full kinase activity. This response occurred through SOCE, as it was blocked by either the SOCE modulator 2-aminoethoxydiphephenyl borate, knockdown of the SOCE molecular component STIM1, or inhibition of Ca2+/calmodulin (CaM)-dependent protein kinase kinase β (CaMKKβ). In thapsigargin-pretreated cells, stimulation of pharmacologically defined M3 mAChRs potentiated SOCE-induced AMPK activation. This potentiation did not involve an increased Ca2+ influx, but was associated with CaM mobilization from membrane to cytosol, increased CaM/CaMKKβ interaction, and enhanced CaMKK stimulation by thapsigargin-induced SOCE. In thapsigargin-pretreated cells Ca2+ re-addition stimulated glucose uptake and increased the membrane expression of the glucose transporter GLUT1. Both responses were significantly potentiated by mAChRs. These data indicate that in human neuroblastoma cells mAChRs up-regulate AMPK and the downstream glucose uptake by triggering not only SOCE but also CaM translocation and enhanced formation of active CaM/CaMKKβ complexes. 相似文献
16.
Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca2+ mobilizing messengers in that its principal function is to mobilize Ca2+ from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca2+ release channel distinct from the well characterized Ca2+ release channels on the (sarco)-endoplasmic reticulum (ER), inositol trisphosphate and ryanodine receptors. In this review,
we discuss the emergence of a novel family of endolysosomal channels, the two-pore channels (TPCs), as likely targets for
NAADP, and how molecular and pharmacological manipulation of these channels is enhancing our understanding of the physiological
roles of NAADP as an intracellular Ca2+ mobilizing messenger. 相似文献
17.
Eugene Emmer Richard P. Rood John H. Wesolek Michael E. Cohen R. Scott Braithwaite Geoffrey W. G. Sharp Heini Murer Mark Donowitz 《The Journal of membrane biology》1989,108(3):207-215
Summary In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl– absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45±13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on theV
max and not on theK
m
for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120nm. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. we conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption. 相似文献
18.
Mads T. S?ndergaard Xixi Tian Yingjie Liu Ruiwu Wang Walter J. Chazin S. R. Wayne Chen Michael T. Overgaard 《The Journal of biological chemistry》2015,290(43):26151-26162
The intracellular Ca2+ sensor calmodulin (CaM) regulates the cardiac Ca2+ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca2+ release. All mutations increased Ca2+ release and rendered RyR2 more susceptible to store overload-induced Ca2+ release (SOICR) by lowering the threshold of store Ca2+ content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca2+ binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca2+ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca2+ binding to the N-domain but markedly decreased the affinity of the C-domain for Ca2+. These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca2+ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca2+-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca2+ concentration, is proposed. 相似文献
19.
Erika Kovacs Judit T��th Be��ta G. V��rtessy K��roly Liliom 《The Journal of biological chemistry》2010,285(3):1799-1808
Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca2+ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca2+-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors. 相似文献
20.
Pei-Feng Chen 《Archives of biochemistry and biophysics》2009,486(2):132-140
Human endothelial nitric-oxide synthase (eNOS) is a complex enzyme, requiring binding of calmodulin (CaM) for electron transfer. The prevailing view is that calcium-activated CaM binds eNOS at the canonical binding site located at residues 493-510, which induces a conformational change to facilitate electron transfer. Here we demonstrated that the CaM enhances the rate of electron transfer from NADPH to FAD on a truncated eNOS FAD subdomain (residues 682-1204) purified from baculovirus-infected Sf9 cells, suggesting more complicated regulatory mechanism of CaM on eNOS. Metabolically 35S-labeled CaM overlay on fusion proteins spanning the entire linear sequence of eNOS revealed three positive 35S-CaM binding fragments: sequence 66-205, sequence 460-592, and sequence 505-759. Synthetic peptides derived from these fragments are tested for their effects on CaM binding and eNOS catalytic activities. Peptides corresponding to the proximal heme-binding site (E1, residues 174-193) and the CD1 linker connecting FAD/FMN subdomains (E4, residues 729-757) bind CaM at both high Ca2+ (Ca2+CaM) and low Ca2+ (apoCaM) concentrations, whereas peptide of the canonical CaM-binding helix (E2, residues 493-510) binds only Ca2+CaM. All three peptides E1, E2 and E4 significantly inhibit oxygenase activity in a concentration-dependent manner, but only E2 effectively inhibits reductase activity. Concurrent experiments with human iNOS showed major differences in the CaM binding properties between eNOS and iNOS. The results suggest that multiple regions of eNOS might interact with CaM with differential Ca2+ sensitivity in vivo. A possible mechanism in regulating eNOS activation and deactivation is proposed. 相似文献