Norma C. Adragna

K-Cl Cotransport: Properties and Molecular Mechanism

K-Cl cotransport (COT), defined first in red blood cells as the Cl-dependent, ouabain-insensitive bidirectional K transport, encoded by at least four KCC (kalium-chloride-cotransport) genes, is now recognized as a functional and structural reality in all cell membranes. As functional system, K-Cl COT is necessary for volume and ionic homeostasis. Since its original discovery by swelling red cells in hyposmotic solutions and by treatment with N-ethylmaleimide (NEM), K-Cl COT has been recognized as one of the prime electroneutral, low ion affinity pathways effecting regulatory volume decrease (RVD). This review first summarizes the general properties of K-Cl COT, including ion dependence, kinetics, thermodynamics and regulation in erythrocytes of various species, and then focuses on the newest findings of the molecular mechanisms behind K-Cl COT, the KCC isoforms and their expression in epithelial cells and in Xenopus oocytes. Based on early biophysical studies on red cells amalgamated with the recent molecular expression studies of the four KCC isoforms, K-Cl COT emerges as one of the oldest membrane transporters that is controlled by a complex redox-dependent cascade of kinases and phosphatases, yet to be defined at the molecular level. Whereas RVD is a primeval role of K-Cl COT for survival of cells challenged by hyposmotic environments, maintenance of intracellular Cl ([Cl]I ) levels away from electrochemical equilibrium and K buffering capability during neuronal function are new additions to the list of physiological functions of this system.

Regulation of K-Cl Cotransport: from Function to Genes

This review intends to summarize the vast literature on K-Cl cotransport (COT) regulation from a functional and genetic viewpoint. Special attention has been given to the signaling pathways involved in the transporter’s regulation found in several tissues and cell types, and more specifically, in vascular smooth muscle cells (VSMCs). The number of publications on K-Cl COT has been steadily increasing since its discovery at the beginning of the 1980s, with red blood cells (RBCs) from different species (human, sheep, dog, rabbit, guinea pig, turkey, duck, frog, rat, mouse, fish, and lamprey) being the most studied model. Other tissues/cell types under study are brain, kidney, epithelia, muscle/smooth muscle, tumor cells, heart, liver, insect cells, endothelial cells, bone, platelets, thymocytes and Leishmania donovani. One of the salient properties of K-Cl-COT is its activation by cell swelling and its participation in the recovery of cell volume, a process known as regulatory volume decrease (RVD). Activation by thiol modification with N-ethylmaleimide (NEM) has spawned investigations on the redox dependence of K-Cl COT, and is used as a positive control for the operation of the system in many tissues and cells. The most accepted model of K-Cl COT regulation proposes protein kinases and phosphatases linked in a chain of phosphorylation/dephosphorylation events. More recent studies include regulatory pathways involving the phosphatidyl inositol/protein kinase C (PKC)-mediated pathway for regulation by lithium (Li) in low-K sheep red blood cells (LK SRBCs), and the nitric oxide (NO)/cGMP/protein kinase G (PKG) pathway as well as the platelet-derived growth factor (PDGF)-mediated mechanism in VSMCs. Studies on VSM transfected cells containing the PKG catalytic domain demonstrated the participation of this enzyme in K-Cl COT regulation. Commonly used vasodilators activate K-Cl COT in a dose-dependent manner through the NO/cGMP/PKG pathway. Interaction between the cotransporter and the cytoskeleton appears to depend on the cellular origin and experimental conditions. Pathophysiologically, K-Cl COT is altered in sickle cell anemia and neuropathies, and it has also been proposed to play a role in blood pressure control. Four closely related human genes code for KCCs (KCC1-4). Although considerable information is accumulating on tissue distribution, function and pathologies associated with the different isoforms, little is known about the genetic regulation of the KCC genes in terms of transcriptional and post-transcriptional regulation. A few reports indicate that the NO/cGMP/PKG signaling pathway regulates KCC1 and KCC3 mRNA expression in VSMCs at the post-transcriptional level. However, the detailed mechanisms of post-transcriptional regulation of KCC genes and of regulation of KCC2 and KCC4 mRNA expression are unknown. The K-Cl COT field is expected to expand further over the next decades, as new isoforms and/or regulatory pathways are discovered and its implication in health and disease is revealed.

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

Erythrocytes play a key role in human and vertebrate metabolism. Tissue O2 supply is regulated by both hemoglobin (Hb)-O2 affinity and erythrocyte rheology, a key determinant of tissue perfusion. Oxygenation-deoxygenation transitions of Hb may lead to re-organization of the cytoskeleton and signalling pathways activation/deactivation in an O2-dependent manner. Deoxygenated Hb binds to the cytoplasmic domain of the anion exchanger band 3, which is anchored to the cytoskeleton, and is considered a major mechanism underlying the oxygenation-dependence of several erythrocyte functions. This work discusses the multiple modes of Hb-cytoskeleton interactions. In addition, it reviews the effects of Mg2+, 2,3-diphosphoglycerate, NO, shear stress and Ca2+, all factors accompanying the oxygenation-deoxygenation cycle in circulating red cells. Due to the extensive literature on the subject, the data discussed here, pertain mainly to human erythrocytes whose O2 affinity is modulated by 2,3-diphosphoglycerate, ectothermic vertebrate erythrocytes that use ATP, and to bird erythrocytes that use inositol pentaphosphate.

Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtained during the last two decades show that spontaneously hypertensive rats, an acceptable experimental model of primary human hypertension, possess increased activity of both ubiquitous and renal cell-specific isoforms of the Na+/H+exchanger (NHE) and Na+-K+-2Cl-cotransporter. Abnormalities of these ion transporters have been found in patients suffering from essential hypertension. Recent genetic studies demonstrate that genes encoding the β- and γ-subunits of ENaC, a renal cell-specific isoform of the Na+-K+-2Cl-cotransporter, and α3-, α1-, and β2-subunits of the Na+-K+pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis of the homology of genome maps, several other genes encoding these transporters, as well as the Na+/H+exchanger and Na+-K+-2Cl-cotransporter, can be predicted in QTL related to the pathogenesis of hypertension. However, despite their location within QTL, analysis of cDNA structure did not reveal any mutation in the coding region of the above-listed transporters in primary hypertension, with the exception of G276L substitution in the α1-Na+-K+pump from Dahl salt-sensitive rats and a higher occurrence of T594M mutation of β-ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities of systems involved in the regulation of their expression and/or function. Further analysis of QTL in F2hybrids of normotensive and hypertensive rats and in affected sibling pairs will allow mapping of genes causing abnormalities of these regulatory pathways.

K-Cl cotransporters, cell volume homeostasis, and neurological disease

K(+)-Cl(-) cotransporters (KCCs) were originally characterized as regulators of red blood cell (RBC) volume. Since then, four distinct KCCs have been cloned, and their importance for volume regulation has been demonstrated in other cell types. Genetic models of certain KCCs, such as KCC3, and their inhibitory WNK-STE20/SPS1-related proline/alanine-rich kinase (SPAK) serine-threonine kinases, have demonstrated the evolutionary necessity of these molecules for nervous system cell volume regulation, structure, and function, and their involvement in neurological disease. The recent characterization of a swelling-activated dephosphorylation mechanism that potently stimulates the KCCs has pinpointed a potentially druggable switch of KCC activity. An improved understanding of WNK/SPAK-mediated KCC cell volume regulation in the nervous system might reveal novel avenues for the treatment of multiple neurological diseases.

Role of nitrite, a nitric oxide derivative, in K-Cl cotransport activation of low-potassium sheep red blood cells.

Abstract. K-Cl cotransport (COT) is the coupled movement of K and Cl, present in most cells, associated with regulatory volume decrease, susceptible to oxidation and functionally overexpressed in sickle cell anemia. The aim of this study was to characterize the effect of the oxidant nitrite (NO2−) on K-Cl COT. NO2− is a stable metabolic end product of the short-lived highly reactive free radical nitric oxide (NO), an oxidant and modulator of ion channels, and a vasodilator. In some systems, the response to NO2− is identical to that of NO. We hypothesized that NO2− activates K-Cl COT. Low potassium (LK) sheep red blood cells (SRBCs) were used as a model. The effect of various concentrations (10−6 to 10−1m) of NaNO2 was studied on K efflux in hypotonic Cl and NO3 media, Cl-dependent K efflux (K-Cl COT), glutathione (GSH), and methemoglobin (MetHb) formation. In support of our hypothesis, K efflux and K-Cl COT were stimulated by increasing concentrations of NaNO2. Stimulation of K efflux was dependent upon external Cl and exhibited a lag phase, consistent with activation of K-Cl COT through a regulatory mechanism. Exposure of LK SRBCs to NaNO2 decreased GSH, an effect characteristic of a thiol-oxidizing agent, and induced MetHb formation. K-Cl COT activity was positively correlated with Methb formation. N-ethyl-maleimide (NEM), a potent activator of K-Cl COT, was used to assess the mechanism of NO2− action. The results suggest that NEM and NO2− utilize at least one common pathway for K-Cl COT activation. Since NaNO2 is also a well known vasodilator, the present findings suggest a role of K-Cl COT in vasodilation.

Incidence of respiratory disorders in neonates born between 34 and 36 weeks of gestation following exposure to antenatal corticosteroids between 24 and 34 weeks of gestation.

We studied the effect of antenatal corticosteroids on the incidence of respiratory disorders in singleton neonates born between 34 and 36 weeks of gestation. Retrospective analysis was conducted of the incidence of respiratory distress syndrome (RDS) and other respiratory disorders (need for mechanical ventilation, continuous positive airway pressure, and prolonged oxygen therapy) among singleton neonates delivered between 34 and 36 weeks of gestation who were exposed to antenatal corticosteroids, compared with neonates who were not exposed. Statistical analyses included two-tailed T tests, two-way analysis of variance for continuous data, and chi-square analysis for ratios. A probability of 0.05 was considered significant. Between January 1, 2000, and December 31, 2004, 1078 neonates were born between 34 and 36 weeks of gestation. Information regarding antenatal corticosteroids was available in 1044: 574 neonates (53.2%) were exposed to antenatal corticosteroids and 470 (43.6%) were not. One thousand and eighteen neonates were admitted to the neonatal intensive care unit. Respiratory disorders were diagnosed in 140 of those exposed to antenatal steroids (24.4%) and in 382 of the nonexposed (81.3%) ( P < 0.0001). Two hundred and ten neonates (20.6%) developed RDS: Of those, 43 were exposed to antenatal corticosteroids and 167 were not (incidence of RDS was 7.5% and 35.5%, respectively; P = 0.0001). The beneficial effects of corticosteroids were similar in both genders. It appears that the exposure of singleton pregnancies to antenatal corticosteroids between 24 and 34 weeks of gestation is associated with a significantly lower incidence of respiratory disorders among neonates born at 34 to 36 weeks of gestation. Further studies are needed to determine whether administering antenatal steroids to women experiencing preterm labor after 34 weeks of gestation would be associated with a similar beneficial effect.

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders, and from Several Other Mammalian Species

Abstract. Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO3 media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO3) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO3 (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity.

K-Cl Cotransport: Immunohistochemical and Ion Flux Studies in Human Embryonic Kidney (HEK293) Cells Transfected with Full-Length and C-Terminal-Domain-Truncated KCC1 cDNAs

Coupled K and Cl movements are mediated by several isoforms of the K-Cl cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C-terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-Cl COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37°C, and the effect of Ctd deletion on K-Cl COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and – CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1-transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluoresence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-Cl COT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly lower in stably and abrogated in transiently –CtdKCC1-transfected cells. Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K-Cl COT in normal and transfected cells. Staurosporine and hydroxylamine were ineffective stimulators. No effect of pH₀ changes (6.3-8.4) was observed in basal or NEM-stimulated K-Cl COT, in both normal and transfected cells. However, inhibition by NEM occurred at pH₀ 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by >30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-Cl COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-Cl COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction.

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.