Sodium and Potassium Requirements for Active Transport of Glutamate by Escherichia coli K-12

Active transport of glutamate by Escherichia coli K-12 requires both Na(+) and K(+) ions. Increasing the concentration of Na(+) in the medium results in a decrease in the K(m) of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K(+). K(+)-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K(+) fully restores their capacity for glutamate uptake when Na(+) ions are also present in the uptake medium. Addition of either K(+) or Na(+) alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K(+) concentration affect the capacity for glutamate uptake but have no effect on the K(m) of the glutamate transport system. Ouabain does not inhibit the (Na(+)-K(+))-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.     

The Wheat cDNA LCT1 Generates Hypersensitivity to Sodium in a Salt-Sensitive Yeast Strain

Salinity affects large areas of agricultural land, and all major crop species are intolerant to high levels of sodium ions. The principal route for Na(+) uptake into plant cells remains to be identified. Non-selective ion channels and high-affinity potassium transporters have emerged as potential pathways for Na(+) entry. A third candidate for Na(+) transport into plant cells is a low-affinity cation transporter represented by the wheat protein LCT1, which is known to be permeable for a wide range of cations when expressed in yeast (Saccharomyces cerevisiae). To investigate the role of LCT1 in salt tolerance we have used the yeast strain G19, which is disrupted in the genes encoding Na(+) export pumps and as a result displays salt sensitivity comparable with wheat. After transformation with LCT1, G19 cells became hypersensitive to NaCl. We show that LCT1 expression results in a strong decrease of intracellular K(+)/Na(+) ratio in G19 cells due to the combined effect of enhanced Na(+) accumulation and loss of intracellular K(+). Na(+) uptake through LCT1 was inhibited by K(+) and Ca(2+) at high concentrations and the addition of these ions rescued growth of LCT1-transformed G19 on saline medium. LCT1 was also shown to mediate the uptake of Li(+) and Cs(+). Expression of two mutant LCT1 cDNAs with N-terminal truncations resulted in decreased Ca(2+) uptake and increased Na(+) tolerance compared with expression of the full-length LCT1. Our findings strongly suggest that LCT1 represents a molecular link between Ca(2+) and Na(+) uptake into plant cells.     

Potassium Transport and the Relationship Between Intracellular Potassium Concentration and Amino Acid Uptake by Cells of a Marine Pseudomonad

Transport of K(+) by K(+)-depleted cells of marine pseudomonad B-16 (ATCC 19855) exhibited saturation kinetics. Rb(+) inhibited both K(+) transport and the K(+)-dependent transport of alpha-aminoisobutyric acid (AIB) into K(+)-depleted cells of the organism in proportion to the concentration of Rb(+) in the suspending medium. Inhibition of the K(+)-dependent uptake of AIB into K(+)-depleted cells by Rb(+) could be overcome by increasing the concentration of K(+) in the medium. When AIB and K(+) were added simultaneously to a suspension of K(+)-depleted cells, the uptake of K(+) occurred immediately and rapidly, whereas the accumulation of AIB occurred only after a lag. The initial uptake rate of AIB was directly proportional to the intracellular K(+) concentration. The intracellular concentration of K(+) and AIB at their steady-state levels increased to a maximum as the Na(+) concentration in the suspending medium was increased. At Na(+) concentrations between 0.2 and 0.3 M, the molar ratio of K(+) to AIB at their intracellular steady-state concentrations was constant at 1.6. At external Na(+) concentrations less than 0.2 M, the cells maintained a relatively higher K(+) intracellular steady-state level than AIB.     

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

上皮细胞K+通道的生理学意义与临床应用:跨上皮细胞转运的驱动力生成和K+循环

K^＋通道在上皮细胞内以极化的方式表达,形成一个庞大的膜蛋白家族。出于对主要依赖Na^＋-K^＋-ATPase而维持的细胞内跨膜K^＋梯度的考虑,K^＋通道在跨上皮细胞转运中的主要作用为：膜电位生成和K^＋循环。本文以肾近端小管和胃壁上皮细胞转运为例简要阐述了K^＋通道的作用。在这两个组织中,K^＋通道活性限速跨上皮细胞转运,调节细胞体积。近年来,药理学工具和转基因动物的实验证实了对K^＋通道的原先认知,并将研究深入到分子水平。K^＋通道的分子结构挑战高亲和力药物分子的设计,及其多组织同时表达的两个典型特征阻碍了高活性、组织特异性小分子治疗的进展。然而,抑制K^＋通道能阻断胃酸分泌等病理生理机制的深入研究,促进K^＋通道药物用于胃病治疗和作为肾脏转运抑制剂用于肾脏相关疾病治疗。     

Identification of K(+) channels in the plasma membrane of maize subsidiary cells

The stomatal complex of Zea mays consists of two guard cells with the pore in between them and two flanking subsidiary cells. Both guard cells and subsidiary cells are important elements for stoma physiology because a well-coordinated transmembrane shuttle transport of potassium and chloride ions occurs between these cells during stomatal movement. To shed light upon the corresponding transport systems from subsidiary cells, subsidiary cell protoplasts were enzymatically isolated and in turn, analyzed with the patch-clamp technique. Thereby, two K(+)-selective channel types were identified in the plasma membrane of subsidiary cells. With regard to their voltage-dependent gating behavior, they may act as hyperpolarization-dependent K(+) uptake and depolarization-activated K(+) release channels during stomatal movement. Interestingly, the K(+) channels from subsidiary cells and guard cells similarly responded to membrane voltage as well as to changes in the K(+) gradient. Further, the inward- and outward-rectifying K(+) current amplitude decreased upon a rise in the intracellular free Ca(2+) level from 2 nM to the micro M-range. The results indicate that the plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K(+) fluxes between these cell types during stomatal movement.     

The contribution of mouse models to our understanding of systemic candidiasis

To maintain optimal intracellular concentrations of alkali-metal-cations, yeast cells use a series of influx and efflux systems. Nonconventional yeast species have at least three different types of efficient transporters that ensure potassium uptake and accumulation in cells. Most of them have Trk uniporters and Hak K(+)-H(+) symporters and a few yeast species also have the rare K(+) (Na(+))-uptake ATPase Acu. To eliminate surplus potassium or toxic sodium cations, various yeast species use highly conserved Nha Na(+) (K(+))/H(+) antiporters and Na(+) (K(+))-efflux Ena ATPases. The potassium-specific yeast Tok1 channel is also highly conserved among various yeast species and its activity is important for the regulation of plasma membrane potential.     

Effects of amphotericin B on ion transport proteins in airway epithelial cells

Topical intranasal application of the antifungal Amphotericin B (AmphoB) has been shown as an effective medical treatment of chronic rhinosinusitis. Because this antibiotic forms channels in lipid membranes, we considered the possibility that it affects the properties and/or cell surface expression of ion channels/pumps, and consequently transepithelial ion transport. Human nasal epithelial cells were exposed apically to AmphoB (50 microM) for 4 h, 5 days (4 h daily), and 4 weeks (4 h daily, 5 days weekly) and allowed to recover for 18-48 h. AmphoB significantly reduced transepithelial potential difference, short-circuit current, and the amiloride-sensitive current. This was not due to generalized cellular toxicity as judged from normal transepithelial resistance and mitochondrial activity, but was related to inhibitory effects of AmphoB on ion transport proteins. Thus, cells exposed to AmphoB for 4 h showed decreased apical epithelial sodium channels (ENaC) activity with no change in basolateral Na(+)K(+)-ATPase activity and K(+) conductance, and reduced amount of alphaENaC, alpha1-Na(+)K(+)-ATPase, and NKCC1 proteins at the cell membrane, but no change in mRNA levels. After a 5-day treatment, there was a significant decrease in Na(+)K(+)-ATPase activity. After a 4-week treatment, a decrease in basolateral K(+) conductance and in alphaENaC and alpha1-Na(+)K(+)-ATPase mRNA levels was also observed. These findings may reflect a feedback mechanism aimed to limit cellular Na(+) overload and K(+) depletion subsequently to formation of AmphoB pores in the cell membrane. Thus, the decreased Na(+) absorption induced by AmphoB resulted from reduced cell surface expression of the ENaC, Na(+)K(+)-ATPase pump and NKCC1 and not from direct inhibition of their activities.     

Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl(-) secretion and increased airway Na(+) absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na(+) transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na(+) channels (ENaC). Here we show that increased airway Na(+) absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na(+) absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease.     

Conservation of Na+ vs. K+ by the rat cortical collecting duct

Regulation of transport by principal cells of the distal nephron contributes to maintenance of Na(+) and K(+) homeostasis. To assess which of these ions is given a higher priority by these cells, we investigated the upregulation of epithelial Na(+) channels (ENaC) in the rat cortical collecting duct (CCD) during Na depletion with and without simultaneous K depletion. ENaC activity, assessed as whole cell amiloride-sensitive current in split-open tubules, was 260 ± 40 pA/cell in K-repleted but virtually undetectable (3 ± 1 pA/cell) in K-depleted animals. This difference was confirmed biochemically by the reduced amounts of the cleaved forms of both the α-ENaC and γ-ENaC subunits measured in immunoblots. In contrast, in K-depleted rats, simultaneously reducing Na intake did not affect the activity of ROMK channels, assessed as tertiapin-Q-sensitive whole cell currents, in the CCDs. The lack of Na current in K-depleted animals was the result of reduced levels of aldosterone in plasma, rather than a reduced sensitivity to the hormone. However, rats on a low-Na, low-K diet for 1 wk did not excrete more Na than those on a low-Na, control-K diet for the same period of time. Immunoblot analysis indicated increased levels of the thiazide-sensitive NaCl cotransporter and the apical Na-H exchanger NHE3. This suggests that with reduced K intake, Na balance is maintained despite reduced aldosterone and Na(+) channel activity by upregulation of Na(+) transport in upstream segments. Under these conditions, Na(+) transport by the aldosterone-sensitive distal nephron is reduced, despite the low-Na intake to minimize K(+) secretion and urinary K losses.     

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.     

Annelid epithelia as models for electrogenic Na+ transport

The electrogenic Na(+) absorption across tight epithelia from invertebrates follows the principles analog to the mechanisms found in vertebrates. Extracellular Na(+)-ions pass the apical cell membranes through highly selective Na(+) channels and follow an electrochemical gradient which is sustained by the basolateral Na(+)/K(+)-ATPases. These apical Na(+) channels are selectively blocked by amiloride and represent the rate-limiting target for the control of transcellular Na(+) uptake. Although annelids express ADH-like peptide hormones, they lack the osmoregulatory mineralocorticoid system with the vertebrate-specific key hormone aldosterone. Thus, their epithelia may represent interesting models for investigation of ion transport regulation. While the formation of urine in the nephridia of, for example, leeches had been subject to intensive studies, the investigation of ion transport across their body wall was largely neglected. We use dissected segments of integuments from the limnic leech Hirudo medicinalis and, recently, from the earthworm Lumbricus terrestris for Ussing chamber experiments. We investigate transintegumental ion transport with focus on control of electrogenic Na(+) uptake and the amiloride-sensitive part of it and identified several extracellular factors as peptide hormones, tri- and divalent cations or purinergic molecules with regulatory effects on it. Meanwhile, there exists a macroscopic view on Na(+) absorption; however, other ion transport mechanisms across annelid integuments still await scientific effort. Here we present a concise synopsis about the electrophysiology of annelid integuments to illustrate the state of science and to evaluate whether further studies in this particular field may be of interest.     

Purinergic stimulation induces Ca2+-dependent activation of Na+-K+-2Cl- cotransporter in human nasal epithelia

Increasing evidence suggests that P2 receptors (P2Rs) in airway epithelial cells perform critical functions in auto- or paracrine regulation of fluid and mucus secretion. In the present study, we characterized the effects of P2R stimulation on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in normal human nasal epithelial (NHNE) cells. [Ca(2+)](i) and pH(i) were measured in primary cultures of NHNE cells using a double perfusion chamber, which enabled us to analyze membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to Na(+)-dependent and bumetanide-sensitive intracellular uptake of NH(4)(+). NKCC activities were observed in the basolateral membrane, but not in the luminal membrane, of NHNE cells. Interestingly, P2Rs were expressed in both membranes, and the stimulation of either luminal or basolateral P2R increased NKCC activity. Blockades of luminal Cl(-) channels, basolateral K(+) channels, or protein kinase C did not affect the activation of NKCC by basolateral P2R stimulation. The effects of luminal P2R stimulation were partially reduced by Cl(-) channel blockers. However, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) treatment completely blocked the stimulatory effects of luminal and basolateral P2Rs on NKCC. In addition, increasing [Ca(2+)](i) by treatment with ionomycin-stimulated NKCC activity. These results provide evidence that stimulation of P2Rs directly activates basolateral NKCC by Ca(2+)-dependent pathways in NHNE cells, which is an important aspect of the purinergic regulation of ion and fluid secretions in human airway epithelia under physiologic and pathologic conditions.     

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.