Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

To identify protein kinases (PK) and phosphatases (PP) involvedin regulation of theNa⁺-K⁺-2Clcotransporter in Ehrlich cells, the effect of various PK and PPinhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculinA (Cal-A) was a potent activator ofNa⁺-K⁺-2Clcotransport (EC₅₀ = 35 nM).Activation by Cal-A was rapid (<1 min) but transient. Inactivation isprobably due to a 10% cell swelling and/or the concurrentincrease in intracellularCl concentration. Cellshrinkage also activates theNa⁺-K⁺-2Clcotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation withCal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH andCa²⁺/calmodulin dependent.Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA byH-89 and KT-5720 inhibited cotransport activity induced by Cal-A and bycell shrinkage, with IC₅₀ values similar to reported inhibition constants of the respective kinases invitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of theNa⁺-K⁺-2Clcotransport activity during regulatory volume increase.

     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV)infection on theNa⁺-K⁺-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]_i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO₃-free media) was reduced inthe absence of external Na⁺ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]_iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa⁺ sensitivities of[Cl]_iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters

Little is known regarding the quaternary structure of cation-Cl- cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl- cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl- cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl- cotransport achieves higher levels of functional diversity than foreseen.     

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Cl- cotransport

The role ofNa⁺-K⁺-2Clcotransport in ion and fluid transport of the corneal endothelium wasexamined by measuring changes in corneal hydration and uptake of⁸⁶Rb by the endothelial celllayer. Isolated, intact rabbit corneas maintain normal hydration whenthey are superfused at the endothelial surface with bicarbonate()-Ringer solutions as aresult of equilibrium between active ion and fluid transport out of thestromal tissue and leak of fluid into stromal tissue from the aqueoushumor. Furosemide and bumetanide did not alter this equilibrium whenthey were added to the superfusion medium. Uptake of⁸⁶Rb by the endothelium of theincubated cornea was increased 25% by bumetanide, but uptake in thepresence of ouabain (70% less than that of controls) was not changedby bumetanide. In Na⁺-free medium,uptake of ⁸⁶Rb was reduced by58%, but it was unchanged inCl-free medium. CalyculinA, a protein phosphatase inhibitor and activator ofNa⁺-K⁺-Clcotransport, was without effect on⁸⁶Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes wassignificantly different when bumetanide was present in the media. It isconcluded thatNa⁺-K⁺-2Clcotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport ofthis cell layer. Its presence in cultured endothelial cells may reflectthe reported importance of this protein in growth, proliferation, anddifferentiation.

     

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.     

Physiology and pathophysiology of Na+/H+ exchange and Na+ -K+ -2Cl- cotransport in the heart, brain, and blood

Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

Furosemide-sensitive Na+-K+ cotransport and cellular metabolism in human erythrocytes

Metabolic depletion of human red cells with 2-deoxy-D-glucose in the presence of EGTA decreased ATP to about 4% of the initial value and increased total ouabain- and furosemide-resistant Na+ and K+ effluxes by 20% and 100%, respectively, and furosemide-sensitive Na+ and K+ effluxes by 100% and 60%, respectively. When ATP was restored, all the components of Na+ and K+ fluxes measured returned to baseline levels suggesting a metabolic dependence.