Differential physiological expression of the invertebrate 2Na+/1H+ antiporter in single epithelial cell type suspensions of lobster hepatopancreas

Lobster (Homarus americanus) hepatopancreas is a complex, heterogeneous tissue composed of four epithelial cell types that individually contribute to the overall functional properties of digestion, absorption, secretion, and detoxification. Previous studies, using purified hepatopancreatic brush border membrane vesicles, have described the properties of an electrogenic, 2Na+/1H+ antiporter in this tissue that regulates the absorption and secretion of these cations. These studies were not able to localize this cation exchange phenomenon to specific epithelial cell types. In the present study, sodium/proton exchange by purified, single cell, suspensions of lobster (Homarus americanus) hepatopancreatic epithelium was investigated using a centrifugal elutriation method to cleanly separate the four individual cell types for subsequent physiological characterization. Results indicate that all four hepatopancreatic epithelial cell types possessed the 2Na+/1H+ antiporter as a result of its unique sigmoidal influx properties. Hill Coefficients, measures of transport sigmodicity obtained from kinetic analyses of 22Na+ influx by single cell type suspensions, varied from 1.56 +/- 0.30 (R-cell suspensions) to 2.79 +/- 0.41 (F-cell suspensions), suggesting that different numbers of sodium ions may be accommodated by each cell type. Both calcium and zinc were competitive inhibitors of 22Na+ influx in E-cells (calcium Ki = 105.1+/-5.2 microM; zinc Ki = 46.2 +/- 7.8 microM), but the extent to which these divalent cations inhibited monovalent cation transport by each cell type varied. It is concluded that different isoforms of the electrogenic 2Na+/1H+ antiporter may be present in each hepatopancreatic cell type and thereby contribute in differing degrees to the cation regulatory functions performed by the overall organ.     

Expression of Na(+) / D-glucose cotransport in Xenopus laevis oocytes by injection of poly(A)(+) RNA isolated from lobster (Homarus americanus) hepatopancreas

Xenopus laevis oocytes were used for expression and characterization of lobster (Homarus americanus) hepatopancreas Na(+)-dependent D-glucose transport activity. Poly(A)(+) RNA from the whole hepatopancreatic tissue was injected and transport activity was assayed by alpha-D-[2-(3)H] glucose. Injection of lobster hepatopancreatic poly(A)(+) RNA resulted in a dose (1-20 ng) and time (1-5 days) dependent increase of Na(+)-dependent D-glucose uptake. Kinetics of Na(+)-dependent glucose transport was a hyperbolic function (K(m)=0.47+/-0.04 mM) of external D-glucose concentration and a sigmoidal function (K(Na)=68.32+/-1.57 mM; Hill coefficient=2.22+/-0.09) of external Na(+) concentration. In addition, Na(+)-dependent D-glucose uptake was significantly inhibited by both (0.1-0.5 mM) phloridzin and (0.1-0.5 mM) methyl-alpha-D-glucopyranoside. After size fractionation through a sucrose density gradient, poly(A)(+) RNA fractions with an average length of 2-4 kb induced a twofold increase in Na(+)-dependent phloridzin-inhibited D-glucose uptake as compared to total poly(A)(+) RNA-induced uptake. The results of this study provide the functional basis to screen lobster hepatopancreatic cDNA libraries for clones encoding putative and still not known crustacean SGLT-type Na(+)/glucose co-transporter(s).     

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.     

DMA增加正常大鼠心肌细胞钙瞬变和收缩

实验观察了钠氢交换或钠钙交换抑制剂 5 (N ,N 二甲基 )氨氯吡咪 (DMA)对正常和心肌肥厚大鼠分离心室肌细胞钙瞬变和细胞收缩的影响。通过负载荧光染料Fura 2 /Am ,应用离子影像分析系统 (IonImagingSystem)同步测定离体大鼠心肌细胞钙瞬变和细胞长度。结果表明 :DMA 10 μmol/L分别使钙瞬变和细胞缩短从对照组的 2 0 9.6 0± 5 4.96和 3.0 7± 0 .97μm增加到 2 38.5 0± 80 .41和 4.0 7± 1.0 2 μm (P <0 .0 5 ,n =7)。应用特异性反向钠钙交换阻断剂KB R7943可完全阻断DMA的激动作用。DMA还可使尼卡地平抑制L 型钙通道后的钙瞬变和细胞收缩增加。在肥厚心肌细胞 ,DMA表现出相同的药理作用 ,但对钙瞬变和细胞缩短的刺激作用更强。结果表明 :DMA可通过反向钠钙交换途径增加正常和肥厚大鼠心肌细胞钙瞬变和细胞收缩 ,且对肥厚心肌细胞的影响比对正常心肌细胞大。     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes.

In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters. The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl. However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations. To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine. The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient. Li(+) and Cs(+) ions were also transported with lower affinity. Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride. Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.     

Biology of the 2Na+/1H+ antiporter in invertebrates

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.     

Catalytic properties of Staphylococcus aureus and Bacillus members of the secondary cation/proton antiporter-3 (Mrp) family are revealed by an optimized assay in an Escherichia coli host

Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary Na(+)(Li(+))/H(+) antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited Na(+)(Li(+))/H(+) antiport. A second paralogue of S. aureus (Mnh2) did not. K(+), Ca(2+), and Mg(2+) did not support significant antiport by any of the test antiporters. All three Na(+)(Li(+))/H(+) Mrp antiporters had alkaline pH optima and apparent K(m) values for Na(+) that are among the lowest reported for bacterial Na(+)/H(+) antiporters. Using a fluorescent probe of the transmembrane electrical potential (DeltaPsi), Mrp Na(+)/H(+) antiport was shown to be DeltaPsi consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher DeltaPsi levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, DeltaPsi-consuming antiporters can elicit increased capacity for DeltaPsi generation in a bacterial host.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.     

Electrophysiological characterization and ionic stoichiometry of the rat brain K(+)-dependent NA(+)/CA(2+) exchanger, NCKX2

We have recently described a novel K(+)-dependent Na(+)/Ca(2+) exchanger, NCKX2, that is abundantly expressed in brain neurons (Tsoi, M., Rhee, K.-H., Bungard, D., Li, X.-F., Lee, S.-L., Auer, R. N., and Lytton, J. (1998) J. Biol. Chem. 273, 4115--4162). The precise role for NCKX2 in neuronal Ca(2+) homeostasis is not yet clearly understood but will depend upon the functional properties of the molecule. Here, we have performed whole-cell patch clamp analysis to characterize cation dependences and ion stoichiometry for rat brain NCKX2, heterologously expressed in HEK293 cells. Outward currents generated by reverse NCKX2 exchange depended on external Ca(2+) with a K(12) of 1.4 or 101 microm without or with 1 mm Mg(2+), and on external K(+) with a K(1/2) of about 12 or 36 mm with choline or Li(+) as counter ion, respectively. Na(+) inhibited outward currents with a K(1/2) of about 60 mm. Inward currents generated by forward NCKX2 exchange depended upon external Na(+) with a K(1/2) of 30 mm and a Hill coefficient of 2.8. K(+) inhibited the inward currents by a maximum of 40%, with a K(1/2) of 2 mm or less, depending upon the conditions. The transport stoichiometry of NCKX2 was determined by observing the change in reversal potential as individual ion gradients were altered. Our data support a stoichiometry for rat brain NCKX2 of 4 Na(+):(1 Ca(2+) + 1 K(+)). These findings provide the first electrophysiological characterization of rat brain NCKX2, and the first evidence that a single recombinantly expressed NCKX polypeptide encodes a K(+)-transporting Na(+)/Ca(2+) exchanger with a transport stoichiometry of 4 Na(+):(1 Ca(2+) + 1 K(+)).     

Ca(2+) removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca(2+)](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na(+)/Ca(2+) exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-D-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca(2+)](i). In addition, in the absence of extracellular Ca(2+), removal of external Na(+) markedly reduced the rate of loss of Ca(2+) from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na(+) on the rate of Ca(2+) removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca(2+) from the cytosol, while PMCA contributes to the other half of the Ca(2+) removal process.