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1.
2.
Tributyltin in the concentration range 1–4μm failed to stimulate Ca2+ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of Pi, despite its promotion of a rapid Cl−/OH− exchange. When 2mm-Pi was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca2+ transport and induced efflux of the ion from the mitochondria in Cl−- or NO3−-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca2+ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca2+ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl−/OH− exchange or producing an oligomycin-like effect. 相似文献
3.
Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment
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Silvia Agostini Elena Chiavacci Marco Matteucci Michele Torelli Letizia Pitto Vincenzo Lionetti 《Journal of cellular and molecular medicine》2015,19(1):227-238
Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2O2-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2−) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2−, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure. 相似文献
4.
Audrey Boniface Claudine Parquet Michel Arthur Dominique Mengin-Lecreulx Didier Blanot 《The Journal of biological chemistry》2009,284(33):21856-21862
Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala4-α-d-Lys3 cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala1(α→α)d-Lys3 and d-Ala4(α→α)d-Lys3 types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)2 cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala1-γ-d-Glu2-X3-d-Ala4-d-Ala5, where X is most often meso-diaminopimelic acid (meso-A2pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala4-d-Ala5 bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala4 and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X3-d-Ala4 bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A2pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link. 相似文献
5.
Stefan Alfred Gross Gustavo Adolfo Guzm��n Ulrich Wissenbach Stephan Ernst Philipp Michael Xi Zhu Dieter Bruns Adolfo Cavali�� 《The Journal of biological chemistry》2009,284(49):34423-34432
TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca2+ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca2+ concentrations (Ca2+i) revealed a dose-dependent activation of TRPC5 channels by internal Ca2+ with EC50 of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca2+i induced by photolysis of caged Ca2+ showed that the Ca2+ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca2+i below 10 μm, suggesting that the action of internal Ca2+ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca2+i above 10 μm. In support of a Ca2+-activation mechanism, the thapsigargin-induced release of Ca2+ from internal stores activated TRPC5 channels transiently, and the subsequent Ca2+ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α1C and β2 subunits of L-type Ca2+ channels, we found that Ca2+ entry through either calcium-release-activated-calcium or voltage-dependent Ca2+ channels is sufficient for TRPC5 channel activation. The Ca2+ entry activated TRPC5 channels under buffering of internal Ca2+ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca2+-activated cation channels that are functionally coupled to Ca2+-selective ion channels through local Ca2+ increases beneath the plasma membrane. 相似文献
6.
Itzhaki I Rapoport S Huber I Mizrahi I Zwi-Dantsis L Arbel G Schiller J Gepstein L 《PloS one》2011,6(4):e18037
Background
The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/Principal Findings
RT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor . U73122Conclusions/Significance
Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release. 相似文献7.
Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides
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Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H+-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-α-d-glucopyranoside, phenyl-α-d-thioglucopyranoside, and phenyl-α-d-4-deoxythioglucopyranoside (PDTGP; Ki = 67, 180, and 327 micromolar, respectively). The Km for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-α-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-α-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-α-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-α-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with Ki values ranging from 600 to 800 micromolar, and phenyl-α-d-thiomannopyranoside, phenyl-β-d-glucopyranoside, and phenylethyl-β-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-α-d-thioglucopyranoside and PDTGP, were transported. 相似文献
8.
Aysha Samad Andrew James James Wong Parini Mankad John Whitehouse Waseema Patel Marta Alves-Simoes Ajith K. Siriwardena Jason I. E. Bruce 《The Journal of biological chemistry》2014,289(34):23582-23595
Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, . These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis. LY294002相似文献
9.
A Steep Dependence of Inward-Rectifying Potassium Channels on
Cytosolic Free Calcium Concentration Increase Evoked by
Hyperpolarization in Guard Cells
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Inactivation of inward-rectifying
K+ channels (IK,in) by a rise in
cytosolic free [Ca2+] ([Ca2+]i)
is a key event leading to solute loss from guard cells and stomatal
closure. However, [Ca2+]i action on
IK,in has never been quantified, nor are its
origins well understood. We used membrane voltage to manipulate
[Ca2+]i (A. Grabov and M.R. Blatt [1998]
Proc Natl Acad Sci USA 95: 4778–4783) while recording
IK,in under a voltage clamp and
[Ca2+]i by Fura-2 fluorescence
ratiophotometry. IK,in inactivation
correlated positively with [Ca2+]i and
indicated a Ki of 329 ± 31
nm with cooperative binding of four Ca2+ ions
per channel. IK,in was promoted by the
Ca2+ channel antagonists Gd3+ and calcicludine,
both of which suppressed the [Ca2+]i rise,
but the [Ca2+]i rise was unaffected by the
K+ channel blocker Cs+. We also found that
ryanodine, an antagonist of intracellular Ca2+ channels
that mediate Ca2+-induced Ca2+ release, blocked
the [Ca2+]i rise, and Mn2+
quenching of Fura-2 fluorescence showed that membrane hyperpolarization
triggered divalent release from intracellular stores. These and
additional results point to a high signal gain in
[Ca2+]i control of
IK,in and to roles for discrete
Ca2+ flux pathways in feedback control of the
K+ channels by membrane voltage.Ca2+ underlies many fundamental regulatory processes
in plants, including adaptive responses to abiotic environmental stress
(Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and
programmed cell death evoked by pathogen attack (Low and Merida, 1996;
Hammondkosack and Jones, 1997). Coordination of changes in
[Ca2+]i and its
integration with downstream response elements are central in coupling
stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell
model, major downstream targets of
[Ca2+]i and their roles
in stomatal function have been identified. Increasing
[Ca2+]i is known to
inactivate IK,in and to activate
Cl− channels, events that bias plasma membrane
transport for net efflux of osmotically active solute and a loss of
turgor, which drives stomatal closure (Blatt and Grabov, 1997).
Furthermore, changes in
[Ca2+]i are associated
with ABA, CO2, and the growth hormone auxin
(Blatt and Grabov, 1997; McAinsh et al., 1997). These
[Ca2+]i signals have been
observed to oscillate (McAinsh et al., 1995; Webb et al., 1996),
characteristics that may constitute “Ca2+
signatures” to encode specific downstream responses (Berridge, 1996).
Yet, despite the evidence for
[Ca2+]i signaling in
guard cells, surprisingly little detail is known about the link between
[Ca2+]i changes and ion
channel activity at the plasma membrane or about the mechanisms
mediating such [Ca2+]i
changes. To our knowledge, in no instance have the characteristics of
ion channel regulation by Ca2+ been quantified
directly in any higher-plant cell.We recently described the coupling of membrane voltage to
[Ca2+]i, demonstrating
that hyperpolarization, whether under a voltage clamp or in the
presence of low [K+]o,
evoked [Ca2+]i increases
in guard cells, and that the voltage threshold for
[Ca2+]i rise was
profoundly altered by ABA (Grabov and Blatt, 1998). Our observations
indicated a link to Ca2+ influx across the plasma
membrane and raised questions about the efficacy of
[Ca2+]i in inactivating
IK,in and about the contributions of
intracellular Ca2+ release to the
[Ca2+]i signal. We have
used membrane voltage to experimentally manipulate
[Ca2+]i and report that
IK,in is strongly dependent on
[Ca2+]i, consistent with
a cooperative binding of four Ca2+ ions to effect
inactivation. Additional experiments indicate that voltage-evoked
[Ca2+]i increases depend
both on Ca2+ influx and on release of
Ca2+ from intracellular stores. These results
underscore the role of
[Ca2+]i as a high-gain
“switch” in the control of IK,in, and
implicate [Ca2+]i in
feedback control linking membrane voltage to the activity of the
K+ channels. 相似文献
10.
The maturation of Ca2+ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca2+ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-Pi in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca2+ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca2+ influx are observed in the presence of 2mm-Pi; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the Pi stimulates further the initial rate of Ca2+ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca2+ for about 50min in the presence of 2mm-Pi. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca2+ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca2+ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca2+ influx assayed in the presence of 2mm-Pi and the time for which mitochondria retain accumulated Ca2+ in the presence of 12.5μm-MgATP and 2mm-Pi. Its administration to neonatal animals leads to an increase in mitochondrial Ca2+ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca2+ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca2+ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver. 相似文献
11.
Hitomi Ichinose Zui Fujimoto Mariko Honda Koichi Harazono Yukifumi Nishimoto Atsuko Uzura Satoshi Kaneko 《The Journal of biological chemistry》2009,284(37):25097-25106
Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a β-l-arabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) α-d-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-β-l-arabinopyranoside was 18 μmol of arabinose/min/mg, which was 67 times higher than that toward p- nitrophenyl-α-d-galactopyranoside. The enzyme could remove 0.1 and 45% l-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel β-domain containing Greek key motifs, another antiparallel β-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of α-d-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of β-l-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of d-galactose bound to α-galactosidase was changed in β-l-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which β-l-arabinopyranosidase is classified as a new member of the GH27 family.Arabinogalactan proteins (AGPs)3 are a family of complex proteoglycans widely distributed in plants (1, 2). AGPs are also found in tree exudate gums and coniferous woods (3) and are characterized by the presence of large amounts of carbohydrate components rich in galactose (all the sugars in the present study are in the d-configuration unless otherwise specified) and l-arabinose and by protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (4). Type II arabinogalactans and short oligosaccharides are the two types of carbohydrates attached to the AGP backbone. Type II arabinogalactans have β-1,3-linked galactosyl backbones in mono- or oligo-β-1,6-galactosyl and/or l-arabinosyl side chains (2, 5). l-Arabinose and lesser amounts of other auxiliary sugars such as glucuronic acid, l-rhamnose, and l-fucose are attached to the side chains primarily at nonreducing termini (2). Molecular and biochemical evidence indicates that AGPs have specific functions during root formation, promotion of somatic embryogenesis, and attraction of pollen tubes to the style (6). However, because many putative protein cores exist and the structures of the carbohydrate moieties are complex, it has been difficult to differentiate one AGP species from another in plant tissues. This, in turn, has made it difficult to assign specific roles to individual AGPs. Despite significant physiological interest in AGPs, there are few studies on glycoside hydrolases that cleave the sugar moieties of these proteins. It is important to study such enzymes because hydrolytic enzymes specific to particular sugar residues or to a type of glycosidic linkage would be useful tools in the structural analysis of AGPs.So far, we have focused on the β-1,3-β-1,6-galactan backbone, which is the common structure of heterogeneous AGPs, to identify glycoside hydrolases acting on AGPs. Galactanases that hydrolyze β-1,3- or β-1,6-galactosyl linkages are useful tools because the enzymes hydrolyze AGPs and produce the constituent carbohydrate moieties of AGPs. We cloned two kinds of galactanases: exo-β-1,3-galactanase (EC 3.2.1.145) from Phanerochaete chrysosporium and endo-β-1,6-galactanase (EC 3.2.1.164) from Trichoderma viride, and demonstrated that the enzymes were novel and could be classified as glycoside hydrolase family 43 (GH43) and family 5 (GH5), respectively (7–9) (see the CAZy website). Genes encoding proteins similar to such enzymes were also identified in the Streptomyces avermitilis genome (10, 11).Because S. avermitilis has two different kinds of galactanases, we focused on finding novel AGP-degrading enzymes. We have cultivated the actinomycete using gum arabic as a carbon source, and isolated a novel β-l-arabinopyranosidase. To the best of our knowledge, the only report on β-l-arabinosidase (EC 3.2.1.88) has been on its purification from Cajanus indicus (12). The amino acid composition of the enzyme was investigated (13), but its sequence remains unknown. In this article, we cloned β-l-arabinopyranosidase from S. avermitilis (SaArap27A), analyzed its catalytic properties, and analyzed the crystal structure of the recombinant enzyme. The results clearly showed that this enzyme is β-l-arabinopyranosidase and is a novel member of the glycoside hydrolase family 27 (GH27). This is the first detailed report on β-l-arabinopyranosidase. 相似文献
12.
13.
Previous studies have shown that the whole-cell current-voltage (I-V) relation of unstimulated sheep parotid cells is dominated by two K+ conductances, one outwardly and the other inwardly rectifying. We now show that once these K+ conductances are blocked by replacement of pipette K+ with Na+ and by the addition of 5 mmol/liter CsCl to the bath, there remains an outwardly rectifying conductance with a reversal potential of 0 mV. Replacement of 120 mmol/liter NaCl in the pipette solution with an equimolar amount of Na-glutamate shifted the reversal potential of this residual current to -55 mV, indicating that the conductance was Cl? selective. The Cl? current was activated by increasing the free Ca2+ in the pipette solution from 10 to 100 nmol/liter. When the Ca2+ concentration in the pipette solution was 10 nmol/liter, the relaxations observed in response to membrane depolarization could be fitted with a single exponential, whose time constant increased from 81 to 183 ms as the pipette potential was increased from -30 to +60 mV. Relaxation analysis showed that the current was activated by membrane depolarization. Reversal potential measurements in experiments in which external Cl? was replaced with various anions, gave the following relative permeabilities: SCN- (1.80) > I- (1.09) > CI- (1) > NO 3 - (0.92) > Br- (0.75). The relative conductances were: SCN- (2.18) > I- (1.07) > Cl? (1.00) > Br- (0.91) > NO 3 - (0.50). The Cl? current was blocked by NPPB (ID50 ≈ 10 μm), DIDS (10 or 30 μmol/liter) and furosemide (100 μmol/liter). 相似文献
14.
Abdirahman M. Jama Jon Fenton Saralili D. Robertson Katalin T?r?k 《The Journal of biological chemistry》2009,284(41):28146-28155
Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is thought to exert its role in memory formation by autonomous Ca2+-independent persistent activity conferred by Thr286 autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca2+] has returned to resting levels. Ca2+ sequestration-induced inhibition, caused by a burst of Thr305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr305/306 sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca2+ withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr305/306 sites. The time courses of Thr305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr305/306 autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca2+ and bound Ca2+/CaM by time courses similar to those seen following Ca2+ sequestration. Our investigation of the activity and mechanisms of phospho-Thr286-αCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca2+ and CaM, allowing a very short, if any, time window for Ca2+/CaM-free phospho-Thr286-αCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr286-αCaMKII (t½ of ∼50 s at 37 °C) suggest a transient kinase activity of ∼1 min duration in the induction of long term potentiation and thus memory formation.Ca2+/calmodulin-dependent protein kinase II (αCaMKII)2 is essential in hippocampal learning and N-methyl-d-aspartate receptor-dependent synaptic plasticity, causing long term potentiation (1, 2). The exact mechanisms of αCaMKII in memory functions have not yet been identified.αCaMKII is a broad specificity Ser/Thr protein kinase, which catalyzes the phosphorylation of over 100 protein and peptide substrates in vitro (3). Uniquely, the CaMKII family possesses two distinct kinase mechanisms. The first mechanism is a “canonical” intrasubunit phosphorylation, commonly found in monomeric kinases, in which the phosphorylatable residue of the substrate bound to the helical subdomain of the catalytic domain at the active site is lined up with the terminal phosphate of ATP (4). Although there is a large number of potential “canonical” substrates for αCaMKII at the synapse (5), so far AMPA receptors have been shown to be possible physiological substrates of αCaMKII (6). For the purpose of this study, syntide 2, a commonly used peptide substrate derived from phosphorylation site 2 of glycogen synthase (7), was chosen.The second mechanism, intersubunit autophosphorylation, takes advantage of the oligomeric organization of CaMKII (8). The most important autophosphorylation site in the α isoform is Thr286, which resides in the vicinity of the autoinhibitory domain (9). Peptide substrates with homologous sequences to this region have been reported to be phosphorylated by αCaMKII. This, however, occurs with a low Vmax, and these substrates show properties of a non-competitive inhibitor with respect to phosphorylation of “canonical” substrates (10) and of Thr286 autophosphorylation itself (11). Examples of such substrates include autocamtide, a peptide substrate derived from the autoinhibitory region (12) and the NR2B subunit of the N-methyl-d-aspartate receptor, which has been identified as a potential physiological target of phospho-Thr286-αCaMKII at the postsynaptic membrane (13). The possible physiological significance of NR2B phosphorylation is not yet known. There is evidence to suggest that Thr286 autophosphorylation is required to achieve full activity of the enzyme, since the unphosphorylatable T286A mutant enzyme has much diminished activity compared with wild type enzyme (14, 15).Thr286 autophosphorylation causes CaM “trapping,” a >104-fold increase in the affinity of αCaMKII for Ca2+/CaM (16–18). At the same time, Thr286 autophosphorylation is also attributed to confer Ca2+- and CaM-independent persistent “autonomous” kinase activity to αCaMKII. However, due to the extremely high affinity of phospho-Thr286-αCaMKII for Ca2+/CaM, [Ca2+] of <10 nm is required to achieve full dissociation of Ca2+/CaM, since CaM trapping occurs by virtue of Ca2+ trapping (19). Partial activity measured upon partial Ca2+ withdrawal therefore may not always reflect Ca2+/CaM-free enzyme (9). Furthermore, the physiological resting [Ca2+] range is 50–100 nm; therefore, phospho-Thr286-αCaMKII is likely always to have residual Ca2+/CaM bound. This may be partially Ca2+-saturated CaM (19).Persistent autonomous activity conferred by Thr286 autophosphorylation is thought to enable αCaMKII to function as a memory molecule (20, 21). In contrast, however, following the development of chemical long term potentiation, rapid inactivation has also been reported (22). The extent of an autonomous activity is further obscured by the finding that Ca2+ sequestration induces a burst of autophosphorylation at residues Thr305/306, followed by a loss of activity (23). Moreover, when examined across a broad range of [Ca2+], the Ca2+/CaM dependence of phospho-Thr286-αCaMKII activity is apparent (19). It is thus vital to establish the mechanisms of activation and inactivation of αCaMKII at the molecular level in order to understand how it may function physiologically in learning and memory. To this end, it is necessary to dissect the mechanisms of Ca2+/CaM dissociation, Thr305/306 autophosphorylation, and inactivation of phospho-Thr286-αCaMKII and to establish the time window for autonomous Ca2+/CaM-independent activity. 相似文献
15.
Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
16.
Weiyi Gong Sandra J Kolker Yuriy Usachev Roxanne Y Walder David L Boyle Gary S Firestein Kathleen A Sluka 《Arthritis research & therapy》2014,16(3):R121
Introduction
Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).Methods
Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca2+]i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca2+]i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.Results
Acidic pH increased [Ca2+]i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca2+]i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca2+]i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca2+]i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.Conclusions
Decreased pH activates ASIC3 resulting in increased [Ca2+]i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca2+]i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage. 相似文献17.
Tetsuji Nakamoto David A. Brown Marcelo A. Catal��n Mireya Gonzalez-Begne Victor G. Romanenko James E. Melvin 《The Journal of biological chemistry》2009,284(8):4815-4822
Salivary glands express multiple isoforms of P2X and P2Y nucleotide
receptors, but their in vivo physiological roles are unclear. P2
receptor agonists induced salivation in an ex vivo submandibular
gland preparation. The nucleotide selectivity sequence of the secretion
response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external
Ca2+ dramatically suppressed the initial ATP-induced fluid
secretion (∼85%). Together, these results suggested that P2X receptors are
the major purinergic receptor subfamily involved in the fluid secretion
process. Mice with targeted disruption of the P2X7 gene
were used to evaluate the role of the P2X7 receptor in
nucleotide-evoked fluid secretion. P2X7 receptor protein and
BzATP-activated inward cation currents were absent, and importantly,
purinergic receptor agonist-stimulated salivation was suppressed by more than
70% in submandibular glands from P2X7-null mice.
Consistent with these observations, the ATP-induced increases in
[Ca2+]i were nearly abolished in
P2X7–/– submandibular acinar and
duct cells. ATP appeared to also act through the P2X7 receptor to
inhibit muscarinic-induced fluid secretion. These results demonstrate that the
ATP-sensitive P2X7 receptor regulates fluid secretion in the mouse
submandibular gland.Salivation is a Ca2+-dependent process
(1,
2) primarily associated with
the neurotransmitters norepinephrine and acetylcholine, release of which
stimulates α-adrenergic and muscarinic receptors, respectively. Both
types of receptors are coupled to G proteins that activate phospholipase
Cβ (PLCβ) during salivary gland stimulation. PLCβ activation
cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and
inositol 1,4,5-trisphosphate (InsP3) production. Activation of
Ca2+-selective InsP3 receptor channels localized to the
endoplasmic reticulum of salivary acinar cells increases the intracellular
free calcium concentration
([Ca2+]i).4
Depletion of the endoplasmic reticulum Ca2+ pool triggers
extracellular Ca2+ influx and a sustained elevation in
[Ca2+]i. This increase in
[Ca2+]i activates Ca2+-dependent
K+ and Cl– channels promoting Cl–
secretion across the apical membrane and a lumen negative, electrochemical
gradient that supports Na+ efflux into the lumen. The accumulation
of NaCl creates an osmotic gradient which drives water movement into the
lumen, thus generating isotonic primary saliva. This primary fluid is then
modified by the ductal system, which reabsorbs NaCl and secretes
KHCO3 producing a final saliva that is hypotonic
(1,
2).Salivation also has a non-cholinergic, non-adrenergic component, the origin
of which is unclear (3). In
addition to muscarinic and α-adrenergic receptors, salivary acinar cells
express other receptors that are coupled to an increase in
[Ca2+]i such as purinergic P2 and substance P
receptors. Like muscarinic and α-adrenergic receptors, P2 receptor
activation leads to a sustained increase in
[Ca2+]i in salivary acinar cells
(4). In contrast, substance P
receptor activation rapidly desensitizes and therefore generates only a
relatively transient increase in [Ca2+]i
(5) that is unlikely to
appreciably contribute to fluid secretion. The purinergic P2 receptor family
is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated
by extracellular di- and triphosphate nucleotides. Activation of both
subfamilies of P2 receptors causes an increase in
[Ca2+]i. P2Y receptors increase
[Ca2+]i via InsP3-induced
Ca2+ mobilization from intracellular stores (similar to
α-adrenergic and muscarinic receptors) while P2X receptors act as
ligand-gated, non-selective cation channels that mediate extracellular
Ca2+ influx (6).
Salivary gland tissues express at least four isoforms of P2X (P2X4
and P2X7) and P2Y (P2Y1 and P2Y2) subtypes;
however, their in vivo physiological significance has yet to be
characterized
(7–11).Our results revealed that ATP acts in isolation to stimulate fluid
secretion from the mouse submandibular gland, but secretion is inhibited when
ATP is simultaneously presented with a muscarinic receptor agonist. Ablation
of the P2X7 gene had no effect on the salivary flow rate
evoked by muscarinic receptor activation, but markedly reduced ATP-mediated
fluid secretion and rescued the inhibitory effects of ATP on muscarinic
receptor activation. Submandibular gland acinar cells from
P2X7–/– animals had dramatically
impaired ATP-activated Ca2+ signaling, consistent with this being
the mechanism responsible for the reduction in ATP-mediated fluid secretion in
these mice. Together, these results demonstrated that ATP regulates
salivation, acting mainly through the P2X7 receptor. Activation of
the P2X7 receptor may play a major role in non-adrenergic,
non-cholinergic stimulated fluid secretion. 相似文献
18.
19.
Internal chloride activity, ai
Cl, and membrane potential, Em, were measured simultaneously in 120 R2 giant neurons of Aplysia californica. ai
Cl was 37.0 ± 0.8 mM, Em was -49.3 ± 0.4 mv, and E
Cl calculated using the Nernst equation was -56.2 ± 0.5 mv. Such values were maintained for as long as 6 hr of continuous recording in untreated neurons. Cooling to 1°–4°C caused ai
Cl to increase at such a rate that 30–80 min after cooling began, E
Cl equalled Em. The two then remained equal for as long as 6 hr. Rewarming to 20°C caused ai
Cl to decline, and E
Cl became more negative than Em once again. Exposure to 100 mM K+-artificial seawater caused a rapid increase of ai
Cl. Upon return to control seawater, ai
Cl declined despite an unfavorable electrochemical gradient and returned to its control values. Therefore, we conclude that chloride is actively transported out of this neuron. The effects of ouabain and 2,4-dinitrophenol were consistent with a partial inhibitory effect. Chloride permeability calculated from net chloride flux using the constant field equation ranged from 4.0 to 36 x 10-8 cm/sec. 相似文献
20.
Nicholas C. Zachos Xuhang Li Olga Kovbasnjuk Boris Hogema Rafiquel Sarker Luke J. Lee Min Li Hugo de Jonge Mark Donowitz 《The Journal of biological chemistry》2009,284(35):23708-23718
Elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca2+ ionophore, 4-bromo- (0.5 μ A23187m): 1) inhibited NHE3 Vmax activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca2+]i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca2+]i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca2+]i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca2+.In normal digestive physiology, the brush border (BB)2 Na+/H+ exchanger, NHE3, mediates the majority of the NaCl and NaHCO3 absorption in the ileum (1). Sequential inhibition and stimulation of NHE3 occur as part of digestive physiology. Short-term regulation of NHE3 activity is achieved through a variety of factors that affect NHE3 turnover number and/or surface expression and often involve a role for the cytoskeleton and accessory proteins, including the multi-PDZ domain containing proteins, NHERF1 and NHERF2 (1, 2). However, many details of this regulation are not understood.The NHERF (Na+/H+ exchanger regulatory factor) family of multi-PDZ domain containing proteins consists of four evolutionarily related members, all of which are expressed in epithelial cells of the mammalian small intestine (2). NHERF1 and NHERF2 have been previously shown to contribute to acute NHE3 stimulation and inhibition (3–10). Recently, two additional PDZ domain containing proteins, termed NHERF3/PDZK1 and NHERF4/PDZK2/IKEPP, have been demonstrated to possess sequence homology with NHERF1 and NHERF2 (11–14). However, unlike NHERF1 and NHERF2, which are comprised of two tandem PDZ domains flanked by a C-terminal ezrin/radixin/moesin binding domain, NHERF3 and NHERF4 consist of four PDZ domains but no other protein-protein interacting domains (12).NHERF3 was initially identified by a yeast two-hybrid screen from a human kidney cDNA library using the membrane-associated protein MAP17, as bait (12). NHERF3 is expressed in the brush border of epithelial cells of the kidney proximal tubule and the small intestine (12). NHERF3 associates with and, in a few cases, has been shown to regulate the activity of multiple apical membrane ion transporters including the cystic fibrosis transmembrane regulator (CFTR), urate anion exchanger 1 (URAT1), sodium-phosphate cotransporter type IIa (NaPiIIa), proton-coupled peptide transporter (PEPT2), and organic cation/carnitine cotransporter (OCTN2) (15–19). Furthermore, NHERF3 directly binds the C terminus of NHE3 (20). Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na+ and Cl− transport. Basal electroneutral sodium absorption was decreased by >40% in the NHERF3 null mouse jejunum (21) and by >80% in the colon (22). In addition, Cinar et al. (22) demonstrated that cAMP and [Ca2+]i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon. However, the mechanism by which NHERF3 regulates NHE3 activity was not resolved.Several physiological and pathophysiological agonists, acting through [Ca2+]i-induced second messenger systems, are known to inhibit electroneutral NaCl absorption in the small intestine (1, 23). Elevation of [Ca2+]i has previously been demonstrated to inhibit NHE3 activity in a NHERF2-, but not NHERF1-dependent manner (5). NHERF2 regulation of NHE3 involves the formation of multiprotein complexes at the plasma membrane that include NHE3, NHERF2, α-actinin-4, and PKCα, which induce endocytic removal of NHE3 from the plasma membrane by a PKC-dependent mechanism (5, 24). Because multiple PDZ proteins exist in the apical pole of epithelial cells (2), the current study was designed to determine whether NHERF3 could reconstitute Ca2+ regulation of NHE3 activity and to define how that occurred. 相似文献