Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Changes in Sodium or Glucose Filtration Rate Modulate Expression of Glucose Transporters in Renal Proximal Tubular Cells of Rat

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na⁺ are potential candidates. In this study we examined the role of glucose and Na⁺ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na⁺-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by ∼36%, SGLT1 by ∼20%, and GLUT2 by ∼100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by ∼100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by ∼150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na⁺ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule. Received: 14 July 2000/Revised: 8 March 2001     

A Single Amino Acid Change Converts the Sugar Sensor SGLT3 into a Sugar Transporter

Background

Sodium-glucose cotransporter proteins (SGLT) belong to the SLC5A family, characterized by the cotransport of Na⁺ with solute. SGLT1 is responsible for intestinal glucose absorption. Until recently the only role described for SGLT proteins was to transport sugar with Na⁺. However, human SGLT3 (hSGLT3) does not transport sugar but causes depolarization of the plasma membrane when expressed in Xenopus oocytes. For this reason SGLT3 was suggested to be a sugar sensor rather than a transporter. Despite 70% amino acid identity between hSGLT3 and hSGLT1, their sugar transport, apparent sugar affinities, and sugar specificity differ greatly. Residue 457 is important for the function of SGLT1 and mutation at this position in hSGLT1 causes glucose-galactose malabsorption. Moreover, the crystal structure of vibrio SGLT reveals that the residue corresponding to 457 interacts directly with the sugar molecule. We thus wondered if this residue could account for some of the functional differences between SGLT1 and SGLT3.

Methodology/Principal Findings

We mutated the glutamate at position 457 in hSGLT3 to glutamine, the amino acid present in all SGLT1 proteins, and characterized the mutant. Surprisingly, we found that E457Q-hSGLT3 transported sugar, had the same stoichiometry as SGLT1, and that the sugar specificity and apparent affinities for most sugars were similar to hSGLT1. We also show that SGLT3 functions as a sugar sensor in a living organism. We expressed hSGLT3 and E457Q-hSGLT3 in C. elegans sensory neurons and found that animals sensed glucose in an hSGLT3-dependent manner.

Conclusions/Significance

In summary, we demonstrate that hSGLT3 functions as a sugar sensor in vivo and that mutating a single amino acid converts this sugar sensor into a sugar transporter similar to SGLT1.     

The Actual Ionic Nature of the Leak Current through the Na/Glucose Cotransporter SGLT1

Expression of the Na⁺/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a “Na⁺ leak” and represents some 5-10% of the maximal Na⁺/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (V_R) of ≈−15 mV as observed for wt SGLT1. V_R showed only a modest negative shift when extracellular Na⁺ concentration ([Na⁺]_o) was lowered and it was completely insensitive to changes in extracellular Cl⁻. When extracellular pH (pH_o) was decreased from 7.5 to 6.5 and 5.5, V_R shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pH_o but other ions must be involved at pH_o 7.5. In the presence of 15 mM [Na⁺]_o (pH_o = 7.5), addition of 75 mM of either Na⁺, Li⁺, Cs⁺, or K⁺ generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter.     

Kinetics of the Reverse Mode of the Na<Superscript>+</Superscript>/Glucose Cotransporter

This study investigates the reverse mode of the Na⁺/glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na⁺- and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The current-voltage relationship saturated at positive membrane voltages (30–50 mV), and approached zero at −150 mV. The half-maximal concentration for αMDG-evoked outward current (K_0.5^αMDG) was 35 mM (at 0 mV). In comparison, K_0.5^αMDG for forward sugar transport was 0.15 mM (at 0 mV). K_0.5^Na was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-O-methyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na⁺/sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.     

Facilitated Lactate Transport by MCT1 when Coexpressed with the Sodium Bicarbonate Cotransporter (NBC) in Xenopus Oocytes

Monocarboxylate transporters (MCT) and sodium-bicarbonate cotransporters (NBC) transport acid/base equivalents and coexist in many epithelial and glial cells. In nervous systems, the electroneutral MCT1 isoform cotransports lactate and other monocarboxylates with H⁺, and is believed to be involved in the shuttling of energy-rich substrates between astrocytes and neurons. The NBC cotransports bicarbonate with sodium and generates a membrane current. We have expressed these transporter proteins, cloned from rat brain (MCT1) and human kidney (NBC), alone and together, by injecting the cRNA into oocytes of the frog Xenopus laevis, and measured intracellular pH changes and membrane currents under voltage-clamp with intracellular microelectrodes, and radiolabeled lactate uptake into the oocytes. We determined the cytosolic buffer capacity, the H⁺ and lactate fluxes as induced by 3 and 10 mM lactate in oocytes expressing MCT1 and/or NBC, and in water-injected oocytes, in salines buffered with 5 mM HEPES alone or with 5% CO₂/10 mM HCO₃⁻ (pH 7.0). In MCT1 + NBC- but not in MCT1- or NBC-expressing oocytes, lactate activated a Na⁺- and HCO₃⁻-dependent membrane current, indicating that lactate/H⁺ cotransport via MCT1, due to the induced pH change, stimulates NBC activity. Lactate/H⁺ cotransport by MCT1 was increased about twofold when MCT1 was expressed together with NBC. Our results suggest that the facilitation of MCT1 transport activity is mainly due to the increase in apparent buffer capacity contributed by the NBC, and thereby suppresses the build-up of intracellular H⁺ during the influx of lactate/H⁺, which would reduce MCT1 activity. Hence these membrane transporters functionally cooperate and are able to increase ion/metabolite transport activity.     

Osmoregulation in the Parasitic Protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K⁺ concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K⁺ concentrations and was inhibited when K⁺ and/or Cl⁻ was absent from the medium. The results suggested that the well-documented Na⁺-K⁺-2Cl⁻ cotransport system was responsible for the K⁺ influx activated during the RVI. However, inhibitors of Na⁺-K⁺-2Cl⁻ cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K⁺ influx in T. foetus.     

Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K⁺/Cl⁻ and the Na⁺/K⁺/2Cl⁻ cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na⁺-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.     

Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi

Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca²⁺ influx and the sustained hyperpolarization is due to H⁺ efflux by activation of the plasma membrane H⁺-ATPase. Protein synthesis is not required for H⁺-ATPase activation. Net K⁺ and Cl⁻ uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl⁻ uptake increases, but net K⁺ flux barely changes and net H⁺ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H⁺-ATPase, and net K⁺ and Cl⁻ uptake during turgor regulation. Other pathways regulating turgor must also exist.     

Light-induced changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H⁺, Ca²⁺, K⁺, and Cl⁻ fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia faba L.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca²⁺ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H⁺, K⁺, and Cl⁻ occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca²⁺ began within the time resolution of our measurements (5 s). In the absence of H⁺ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO₂ uptake by photosynthesizing tissue, whereas activation of the plasma membrane H⁺ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO₂ diffusion into the leaf.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

Shoot Na+ Exclusion and Increased Salinity Tolerance Engineered by Cell Type–Specific Alteration of Na+ Transport in Arabidopsis

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na⁺ toxicity of many crop plants is correlated with overaccumulation of Na⁺ in the shoot. We have previously suggested that the engineering of Na⁺ exclusion from the shoot could be achieved through an alteration of plasma membrane Na⁺ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na⁺ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na⁺ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na⁺ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na⁺. Plants with reduced shoot Na⁺ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na⁺ and grew poorly. Our results demonstrate that the modification of a specific Na⁺ transport process in specific cell types can reduce shoot Na⁺ accumulation, an important component of salinity tolerance of many higher plants.     

Ionic components of electric current at rat corneal wounds

Background

Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents?

Methodology/Principal Findings

Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca²⁺ efflux increased steadily whereas K⁺ showed an initial large efflux which rapidly decreased. Surprisingly, Na⁺ flux at wounds was inward. A most significant observation was a persistent large influx of Cl⁻, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl⁻, Na⁺ and K⁺. Injury to the cornea caused significant changes in distribution and expression of Cl⁻ channel CLC2.

Conclusions/Significance

These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca²⁺ and Cl⁻ fluxes appear to be mainly actively regulated, while K⁺ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.     

Trinucleotide Insertions, Deletions, and Point Mutations in Glucose Transporters Confer K+ Uptake in Saccharomyces cerevisiae

Deletion of TRK1 and TRK2 abolishes high-affinity K⁺ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1Δ trk2Δ cells on K⁺-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2. ⁸⁶Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K⁺, the mutant hexose transporters also conferred permeation of other cations, including Na⁺. Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence.     

Voltage clamp fluorometric measurements on a type II Na+-coupled Pi cotransporter: shedding light on substrate binding order

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na⁺-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na⁺) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na⁺ ions precede P_i binding, and only the second Na⁺ binding transition is voltage dependent. Moreover, we show that Li⁺, which does not drive cotransport, interacts with the first Na⁺ binding transition. The results were incorporated in a new model of the transport cycle of type II Na⁺/P_i cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.     

A Saccharomyces cerevisiae Mutant Lacking a K+/H+ Exchanger

The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K⁺/H⁺ antiporter (873 amino acids) in Saccharomyces cerevisiae was disrupted by homologous recombination. The core protein is similar to the putative Na⁺/H⁺ antiporters from Enterococcus hirae (NAPA gene) and Lactococcus lactis (LLUPP gene) and the putative K⁺/H⁺ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1 gene resulted in an increased K⁺ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of ⁸⁶Rb⁺ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K⁺ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na⁺]_in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H⁺) or sodium ions (Na⁺), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H⁺- and Na⁺-driven components that functions as a sodium motor. Changing external sodium concentration ([Na⁺]_ex) in the range 1–85 mM resulted in changes in [Na⁺]_in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na⁺]_in and [Na⁺]_ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.     

Alkali Metal Cation Transport and Homeostasis in Yeasts

Summary: The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K⁺ and Na⁺, is of utmost importance for living cells, since they determine cell volume, intracellular pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K⁺ transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na⁺ can be tolerated due to the existence of an Na⁺, K⁺-ATPase and an Na⁺, K⁺/H⁺-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.