共查询到20条相似文献,搜索用时 15 毫秒
1.
Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant
Moshe Giladi Su Youn Lee Bosmat Refaeli Reuben Hiller Ka Young Chung Daniel Khananshvili 《BBA》2019,1860(3):189-200
The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na+ and Ca2+, whereas NCLX can exchange Ca2+ with either Na+ or Li+. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (Kint?=?0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint?=?0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca2+-site” (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na+/Li+ (but not for Ca2+) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca2+ homeostasis in distinct sub-cellular compartments. 相似文献
2.
《Cell calcium》2018
Prokaryotic and eukaryotic Na+/Ca2+ exchangers (NCX) control Ca2+ homeostasis. NCX orthologs exhibit up to 104-fold differences in their turnover rates (kcat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) Km values (Kint = KmCyt/KmExt) are highly asymmetric and alike (Kint ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in kcat at comparable Kint remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on kcat and Kint. Among 55 tested residues, 26 mutations affect either kcat or Kint, where two major groups can be distinguished. The first group of mutations (14 residues) affect kcat rather than Kint. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect Kint rather than kcat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca2+ and Na+ signaling. 相似文献
3.
Maria Barthmes Jun Liao Youxing Jiang Andrea Brüggemann Christian Wahl-Schott 《The Journal of general physiology》2016,147(6):485-496
Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology. 相似文献
4.
《Cell calcium》2020
Structure-dynamic analysis of archaeal NCX (NCX_Mj) provided new insights into the underlying mechanisms of ion selectivity, ion-coupled alternating access, ion occlusion, and transport catalysis. This knowledge is relevant, not only for prokaryotic and eukaryotic NCXs, but also for other families belonging to the superfamily of Ca2+/CA antiporters. In parallel with the ion transport mechanisms, the structure-dynamic determinants of regulatory CBD1 and CBD2 domains have been resolved according to which the Ca2+-induced allosteric signal is decoded at the two-domain interface and "secondarily" modified by a splicing segment at CBD2. The exon-dependent combinations within the splicing segment control the number of Ca2+ binding sites (from zero to three) at CBD2, as well as the Ca2+ binding affinity and Ca2+ off-rates at both CBDs. The exon-dependent combinations specifically rigidify the local segments at CBDs, yielding the Ca2+-dependent activation (through Ca2+ binding to CBD1) and Ca2+-dependent alleviation of Na+-induced inactivation (through Ca2+ binding with CBD2). The exon-dependent synergistic interactions between CBDs characteristically differ in NCX1 and NCX3, thereby underscoring the physiological relevance of structure-controlled shaping of ion-dependent regulation in tissue-specific NCX variants. How the ion-dependent regulatory modules operate in conjunction with other regulators (PIP2, palmitoylation, XIP, among the others) of NCX is an open question that remains to be determined. 相似文献
5.
Dynamic features of Ca2+ interactions with transport and regulatory sites control the Ca2+-fluxes in mammalian Na+/Ca2+(NCX) exchangers bearing the Ca2+-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na+/Ca2+ exchange (kcat = 0.5 ± 0.2s−1) in WT–NCX_Mj is 103–104 times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (Kint) for bidirectional Ca2+ movements (defined as the ratio between the cytosolic and extracellular Km of Ca2+/Ca2+ exchange) is asymmetric, Kint = 0.15 ± 0.5. Therefore, the Ca2+ movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr Km values having a physiological relevance. Bidirectional Ca2+ movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (kcat) ∼10 fold, while increasing the Kint values 100–250-fold (Kint = 15–35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca2+ movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (Kint) of bidirectional Ca2+ movements seems to be comparable among evolutionary diverged NCX variants. 相似文献
6.
《Cell calcium》2020
K+-dependent Na+/Ca2+ exchanger proteins (NCKX1-5) of the SLC24 gene family play important roles in a wide range of biological processes including but not limited to rod and cone photoreceptor vision, olfaction, enamel formation and skin pigmentation. NCKX proteins are also widely expressed throughout the brain and NCKX2 and NCKX4 knockouts in mice have specific phenotypes. Here we review our work on structure-function relationships of NCKX proteins. We discuss membrane topology, domains critical to transport function, and residues critical to cation binding and transport function, all in the context of crystal structures that were obtained for the archaeal Na+/Ca2+ exchanger NCX_Mj. 相似文献
7.
8.
《Cell calcium》2018
K+-dependent Na+-Ca2+ exchangers (NCKXs) play an important role in Ca2+ homeostasis in many tissues. NCKX proteins are bi-directional plasma membrane Ca2+-transporters which utilize the inward Na+ and outward K+ gradients to move Ca2+ ions into and out of the cytosol (4Na+:1Ca2+ + 1 K+). In this study, we carried out scanning mutagenesis of all the residues of the highly conserved α-1 and α-2 repeats of NCKX2 to identify residues important for K+ transport. These structural elements are thought to be critical for cation transport. Using fluorescent intracellular Ca2+-indicating dyes, we measured the K+ dependence of transport carried out by wildtype or mutant NCKX2 proteins expressed in HEK293 cells and analyzed shifts in the apparent binding affinity (Km) of mutant proteins in comparison with the wildtype exchanger. Of the 93 residue substitutions tested, 34 were found to show a significant shift in the external K+ ion dependence of which 16 showed an increased affinity to K+ ions and 18 showed a decreased affinity and hence are believed to be important for K+ ion binding and transport. We also identified 8 residue substitutions that resulted in a partial loss of K+ dependence. Our biochemical data provide strong support for the cation binding sites identified in a homology model of NCKX2 based on crystal structures reported for distantly related archaeal Na+-Ca2+ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca2+ and Na+ binding. Supported by CIHR MOP-81327. 相似文献
9.
Na+/Ca2+ exchangers (NCXs) promote the extrusion of intracellular Ca2+ to terminate numerous Ca2+-mediated signaling processes. Ca2+ interaction at two Ca2+ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca2+ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca2+ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca2+ binding. The carboxylate residues that coordinate two Ca2+ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca2+ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca2+ binding site to change Ca2+ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca2+ regulatory behavior. 相似文献
10.
Michela Ottolia Debora A. Nicoll Kenneth D. Philipson 《The Journal of biological chemistry》2009,284(47):32735-32741
We expressed full-length Na+-Ca2+ exchangers (NCXs) with mutations in two Ca2+-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca2+-dependent regulation of NCX. CBD1 has four Ca2+-binding sites, and mutation of residues Asp421 and Glu451, which primarily coordinate Ca2+ at sites 1 and 2, had little effect on regulation of NCX by Ca2+. In contrast, mutations at residues Glu385, Asp446, Asp447, and Asp500, which coordinate Ca2+ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca2+. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca2+ affinity but retained regulation, confirming a contribution of CBD2 to Ca2+ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca2+ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca2+-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca2+-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca2+, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca2+-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca2+, although CBD2 is also necessary for Ca2+-dependent regulation. 相似文献
11.
Vincent C. K. Chiu Donald Mouring Duncan H. Haynes 《Journal of bioenergetics and biomembranes》1983,15(1):13-25
The effect of Hg2+ and Ch3-Hg+ on the passive and active transport properties of the Ca2+-Mg2+-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10–5 and 10–4
M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca2+. The mercurials increase the passive Ca2+ and Mg2+ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca2+-Mg2+-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K+ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca2+-Mg2+-ATPase and other SR proteins in black lipid membranes, as presented by others. 相似文献
12.
《Biophysical journal》2021,120(17):3664-3675
Na+/Ca2+ exchangers (NCXs) are secondary active transporters that couple the translocation of Na+ with the transport of Ca2+ in the opposite direction. The exchanger is an essential Ca2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca2+-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca2+ sensor called CBD12. Binding of intracellular Ca2+ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca2+-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR (1H-15N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca2+-bound state is less frequently sampled. These results are consistent with the view that Ca2+ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs’ allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems. 相似文献
13.
After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg2+ than by Ca2+ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca2+ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca2+-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg2+ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.
Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca2+ ions, implying that the selective Ca2+ effect on photosystem II (selective Ca2+ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.
相似文献14.
Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. Structure/function analysis of the Arabidopsis H+/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca2+ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca2+ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca2+ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca2+ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca2+ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca2+ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca2+ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues. 相似文献
15.
Sumio Ishijima Zenpei Shigemi Hiroaki Adachi Nana Makinouchi Ikuko Sagami 《生物化学与生物物理学报:生物膜》2012,1818(9):2202-2208
Magnesium (Mg2+) plays critical role in many physiological processes. The mechanism of Mg2+ transport has been well documented in bacteria; however, less is known about Mg2+ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg2+ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg2+ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg2+ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg2+ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg2+ uptake. The AtMRS2-10-mediated Mg2+ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co2+ and Ni2+; however, it was not inhibited by Ca2+, Fe2+, or Fe3+. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al3+. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships. 相似文献
16.
Benoît Roux 《Biophysical journal》2010,98(12):2877-2885
The ability to discriminate between different cations efficiently is essential for the proper physiological functioning of many membrane transport proteins. One obvious mechanism of ion selectivity is when a binding site is structurally constrained by the protein architecture and its geometry is precisely adapted to fit an ion of a given size. This mechanism is not effective in the case of flexible protein binding sites that are able to deform structurally or to adapt to a bound ion. In this study, the concept of nontrivial ion selectivity arising in a highly flexible protein binding site conceptually represented as a microdroplet of ligands confined to a small volume is explored. The environment imposed by the spatial confinement is a critical feature of the reduced models. A large number of reduced binding site models (1077) comprising typical ion-coordinating ligands (carbonyl, hydroxyl, carboxylate, water) are constructed and characterized for Na+/K+ and Ca2+/Ba2+ size selectivity using free energy perturbation molecular dynamics simulations. Free energies are highly correlated with the sum of ion-ligand and ligand-ligand mean interactions, but the relative balance of those two contributions is different for K+-selective and Na+-selective binding sites. The analysis indicates that both the number and the type of ligands are important factors in ion selectivity. 相似文献
17.
《生物化学与生物物理学报:生物膜》2018,1860(11):2184-2191
Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg2+ uptake into proteoliposomes. The rapid Mg2+ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni2+ transport activity but almost no Co2+ transport activity. The rapid Mg2+ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg2+ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg2+ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al. 相似文献
18.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,889(2):225-235
Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin. 相似文献
19.
Sulfate Transport in Cultured Tobacco Cells : EFFECTS OF CALCIUM AND SULFATE CONCENTRATION 总被引:5,自引:2,他引:3
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sulfate transport by tobacco cells (var. Xanthi) cultured in liquid medium was investigated. Monophasic uptake was observed over a sulfate concentration range from 0.01 to 10 millimolar, and the Km was 20 micromolar. A time-dependent stimulation of transport was observed when cells were incubated in medium containing 0.5 millimolar Ca2+. Calcium stimulation was dependent on the culture cycle and was maximal during the early exponential phase. It was not observed in sulfur-deficient cells with high transport rates and was relatively small in sulfate-loaded cells with low transport rates. A kinetic analysis showed that Ca2+ increased the maximum rate of transport without affecting the Km. 相似文献
20.
Patrick C.L. Wong James C.K. Lai Louis Lim Alan N. Davison 《Journal of inorganic biochemistry》1981,14(3):253-260
AlCl3, MnCl2, and CdCl2 inhibited the rates of accumulation of 14C] L-glutamate and 3H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC50) of GABA transport were 316 μM, 7.4 mM, and 1.4 mM, respectively. Ca2+ (1 mM) enhanced the inhibitory effect of Al3+ (IC50 decreased to 149 μM) but antagonized that of Mn2+ (IC50 = 10 mM) and Cd2+ (IC50 = 2.1 mM). For glutamate transport 1 mM Ca2+ changed the IC50 values from 299 to 224 μm for Al3+, 7.1 to 10 mM for Mn2+, and 2 to 3 mM for Cd2+. In contrast, the rates of accumulation of 14C] 2-deoxy-glucose and 3H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al3+, Mn2+, and Cd2+ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane. 相似文献