Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes

Background Halophytes are the flora of saline soils. They adjust osmotically to soil salinity by accumulating ions and sequestering the vast majority of these (generally Na⁺ and Cl⁻) in vacuoles, while in the cytoplasm organic solutes are accumulated to prevent adverse effects on metabolism. At high salinities, however, growth is inhibited. Possible causes are: toxicity to metabolism of Na⁺ and/or Cl⁻ in the cytoplasm; insufficient osmotic adjustment resulting in reduced net photosynthesis because of stomatal closure; reduced turgor for expansion growth; adverse cellular water relations if ions build up in the apoplast (cell walls) of leaves; diversion of energy needed to maintain solute homeostasis; sub-optimal levels of K⁺ (or other mineral nutrients) required for maintaining enzyme activities; possible damage from reactive oxygen species; or changes in hormonal concentrations.Scope This review discusses the evidence for Na⁺ and Cl⁻ toxicity and the concept of tissue tolerance in relation to halophytes.Conclusions The data reviewed here suggest that halophytes tolerate cytoplasmic Na⁺ and Cl⁻ concentrations of 100–200 mm, but whether these ions ever reach toxic concentrations that inhibit metabolism in the cytoplasm or cause death is unknown. Measurements of ion concentrations in the cytosol of various cell types for contrasting species and growth conditions are needed. Future work should also focus on the properties of the tonoplast that enable ion accumulation and prevent ion leakage, such as the special properties of ion transporters and of the lipids that determine membrane permeability.     

Cl? and K+ channels and their role in primary brain tumour biology

Profound cell volume changes occur in primary brain tumours as they proliferate, invade surrounding tissue or undergo apoptosis. These volume changes are regulated by the flux of Cl⁻ and K⁺ ions and concomitant movement of water across the membrane, making ion channels pivotal to tumour biology. We discuss which specific Cl⁻ and K⁺ channels are involved in defined aspects of glioma biology and how these channels are regulated. Cl⁻ is accumulated to unusually high concentrations in gliomas by the activity of the NKCC1 transporter and serves as an osmolyte and energetic driving force for volume changes. Cell volume condensation is required as cells enter M phase of the cell cycle and this pre-mitotic condensation is caused by channel-mediated ion efflux. Similarly, Cl⁻ and K⁺ channels dynamically regulate volume in invading glioma cells allowing them to adjust to small extracellular brain spaces. Finally, cell condensation is a hallmark of apoptosis and requires the concerted activation of Cl⁻ and Ca²⁺-activated K⁺ channels. Given the frequency of mutation and high importance of ion channels in tumour biology, the opportunity exists to target them for treatment.     

Immunolocalization of Na/K–ATPase and Na/K/2Cl cotransporter in the tubular epithelia of sea snake salt glands

The sublingual salt gland is the primary site of salt excretion in sea snakes; however, little is known about the mechanisms mediating ion excretion. Na⁺/K⁺–ATPase (NKA) and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) are two proteins known to regulate membrane potential and drive salt secretion in most vertebrate secretory cells. We hypothesized that NKA and NKCC would localize to the basolateral membranes of the principal cells comprising the tubular epithelia of sea snake salt glands. Although there is evidence of NKA activity in salt glands from several species of sea snake, the localization of NKA and NKCC and other potential ion transporters remains unstudied. Using histology and immunohistochemistry, we localized NKA and NKCC in salt glands from three species of laticaudine sea snake: Laticauda semifasciata, L. laticaudata, and L. colubrina. Antibody specificity was confirmed using Western blots. The compound tubular glands of all three species were found to be composed of serous secretory epithelia, and NKA and NKCC were abundant in the basolateral membranes. These results are consistent with the morphology of secretory epithelia found in the rectal salt glands of marine elasmobranchs, the nasal glands of marine birds and the gills of teleost fishes, suggesting a similar function in regulating ion secretion.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl^- with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl^- cotransporter NKCC1 and the K⁺/Cl^- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

Kidney ion handling genes and their interaction in blood pressure control

Hypertension affects 30% of adults and is the leading risk factor for cardiovascular disease. Kidney sodium reabsorption plays a vital role in the initial stage and development of essential hypertension. It has been extensively reported that the variants of kidney ion handling genes are associated to blood pressure, and clinical features of hypertension. However, the underlying mechanisms by which these variants alter protein function are rarely summarized. In addition, the variation of one single gene is often limited to induce a significant effect on blood pressure. In the past few decades, the influence by genes × genes (G × G) and/or genotype × environment (G × E) interactions on a given trait, for example, blood pressure, have been widely considered, especially in studies on polygenic genetic traits. In the present review, we discuss the progress in genetics studies on kidney ion handling genes, encoding Na⁺ channels (Na⁺-Cl⁻ cotransporter [NCC], Na-K-2Cl cotransporter [NKCC2], epithelial Na⁺ channels [ENaCs]), K⁺ channel (renal outer medullary potassium channel [ROMK]), and Cl⁻ channels (Pendrin, chloride voltage-gated channel Kb [CLC-Kb]), respectively, and their upstream kinases, WNKs and SGK1. We seek to clarify how these genes are involved in kidney sodium absorption and influence blood pressure, especially emphasizing the underlying mechanisms by which genetic variants alter protein functions and interaction in blood pressure regulation. The present review aims to enhance our understanding of the important role of kidney ion handling genes/channels in blood pressure control.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Compartments and Fluxes of K, NA, and CL in Avena Coleoptile Cells

By the compartmental analysis method of MacRobbie and Dainty, and Pitman, estimates of K⁺, Na⁺, and Cl⁻ concentrations and fluxes were obtained for the cytoplasm and vacuole of coleoptile cells of oat, Avena sativa L. cv. Victory. Double labeling was used in experiments with ⁴²K plus ²²Na and with ⁴²K plus ³⁶Cl in a complete nutrient solution. At the plasmalemma, according to the Ussing-Teorell flux ratio equation, Na⁺ is pumped out and Cl⁻ is actively transported inward. The results with K⁺ are less conclusive, but it is probably pumped in. At the tonoplast there is an active inward transport of Na⁺ and probably of K⁺, but the status of Cl⁻ is uncertain, depending upon whether there is an electrical potential difference between the cytoplasm and vacuole. The results suggest that ion selectivity resides mostly in the plasmalemma. Possible errors in the estimates and interpretations are discussed.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Functional Expression of Human NKCC1 from a Synthetic Cassette-Based cDNA: Introduction of Extracellular Epitope Tags and Removal of Cysteines

The Na-K-Cl cotransporter (NKCC) couples the movement of Na⁺, K⁺, and Cl⁻ ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl⁻ -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully “cys-less” NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.     

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na⁺, H⁺ (OH⁻), bicarbonate, borate, and NH₄⁺. Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH₄⁺, Na⁺, and H⁺ contributions to electrogenic ion transport through SLC4A11 stably expressed in Na⁺/H⁺ exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH₄Cl were determined by the [NH₃]_o, not [NH₄⁺]_o, and current amplitudes varied with the [H⁺] gradient. These currents were relatively unaffected by removal of Na⁺, K⁺, or Cl⁻ from the bath but could be reduced by inclusion of NH₄Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH₄Cl. Reversal potential shifts in response to changing [NH₃]_o and pH_o suggested an NH₃/H⁺-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH₃ but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH₃/H⁺). NH₃-dependent currents were insensitive to 10 μm ethyl-isopropyl amiloride or 100 μm 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid. We propose that SLC4A11 is an NH₃/2H⁺ co-transporter exhibiting unique characteristics.     

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.     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Molecular mechanisms of potassium and sodium uptake in plants

Potassium (K⁺) is an essential nutrient and the most abundant cation in plants, whereas the closely related ion sodium (Na⁺) is toxic to most plants at high millimolar concentrations. K⁺ deficiency and Na⁺ toxicity are both major constraints to crop production worldwide. K⁺ counteracts Na⁺ stress, while Na⁺, in turn, can to a certain degree alleviate K⁺ deficiency. Elucidation of the molecular mechanisms of K⁺ and Na⁺ transport is pivotal to the understanding – and eventually engineering – of plant K⁺ nutrition and Na⁺ sensitivity. Here we provide an overview on plant K⁺ transporters with particular emphasis on root K⁺ and Na⁺ uptake. Plant K⁺-permeable cation transporters comprise seven families: Shaker-type K⁺ channels, `two-pore' K⁺ channels, cyclic-nucleotide-gated channels, putative K⁺/H⁺ antiporters, KUP/HAK/KT transporters, HKT transporters, and LCT1. Candidate genes for Na⁺ transport are the KUP/HAK/KTs, HKTs, CNGCs, and LCT1. Expression in heterologous systems, localization in plants, and genetic disruption in plants will provide insight into the roles of transporter genes in K⁺ nutrition and Na⁺ toxicity.     

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl^? through the transporters including the Na⁺, K⁺-ATPase (NKA), Na⁺, K⁺, 2Cl^? cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl^? secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (> 1 day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24 h, the Cl^? secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.