Pharmacology of Compounds Targeting Cation-Chloride Cotransporter Physiology.

Transporters of the solute carrier family 12 (SLC12) carry inorganic cations such as Na+ and/or K+ alongside Cl across the plasma membrane of cells. These tightly coupled, electroneutral, transporters are expressed in almost all tissues/organs in the body where they fulfil many critical functions. The family includes two key transporters participating in salt reabsorption in the kidney: the Na-K-2Cl cotransporter-2 (NKCC2), expressed in the loop of Henle, and the Na-Cl cotransporter (NCC), expressed in the distal convoluted tubule. NCC and NKCC2 are the targets of thiazides and "loop" diuretics, respectively, drugs that are widely used in clinical medicine to treat hypertension and edema. Bumetanide, in addition to its effect as a loop diuretic, has recently received increasing attention as a possible therapeutic agent for neurodevelopmental disorders. This chapter also describes how over the past two decades, the pharmacology of Na+ independent transporters has expanded significantly to provide novel tools for research. This work has indeed led to the identification of compounds that are 100-fold to 1000-fold more potent than furosemide, the first described inhibitor of K-Cl cotransport, and identified compounds that possibly directly stimulate the function of the K-Cl cotransporter. Finally, the recent cryo-electron microscopy revolution has begun providing answers as to where and how pharmacological agents bind to and affect the function of the transporters.

Na/H Exchange Regulatory Factor 1 Deficient Mice Show Evidence of Oxidative Stress and Altered Cisplatin Pharmacokinetics.

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2-4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

Phosphate Transport in Epithelial and Nonepithelial Tissue.

Phosphate is an essential nutrient for life and is a critical component of bone formation, a major signaling molecule, and structural component of cell walls. Phosphate is also a component of high-energy compounds (i.e., AMP, ADP, and ATP) and essential for nucleic acid helical structure (i.e., RNA and DNA). Phosphate plays a central role in the process of mineralization, normal serum levels being associated with appropriate bone mineralization, while high and low serum levels are associated with soft tissue calcification. The serum concentration of phosphate and the total body content of phosphate are highly regulated, a process that is accomplished by the coordinated effort of two families of sodium-dependent transporter proteins. The three isoforms of the SLC34 family (SLC34A1-A3) show very restricted tissue expression and regulate intestinal absorption and renal excretion of phosphate. SLC34A2 also regulates the phosphate concentration in multiple lumen fluids including milk, saliva, pancreatic fluid, and surfactant. Both isoforms of the SLC20 family exhibit ubiquitous expression (with some variation as to which one or both are expressed), are regulated by ambient phosphate, and likely serve the phosphate needs of the individual cell. These proteins exhibit similarities to phosphate transporters in nonmammalian organisms. The proteins are nonredundant as mutations in each yield unique clinical presentations. Further research is essential to understand the function, regulation, and coordination of the various phosphate transporters, both the ones described in this review and the phosphate transporters involved in intracellular transport.

PBT2 acts through a different mechanism of action than other 8-hydroxyquinolines: an X-ray fluorescence imaging study.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

NHERF1 Loss Upregulates Enzymes of the Pentose Phosphate Pathway in Kidney Cortex.

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

Sodium Transporters in Human Health and Disease.

Sodium (Na+) electrochemical gradients established by Na+/K+ ATPase activity drives the transport of ions, minerals, and sugars in both excitable and non-excitable cells. Na+-dependent transporters can move these solutes in the same direction (cotransport) or in opposite directions (exchanger) across both the apical and basolateral plasma membranes of polarized epithelia. In addition to maintaining physiological homeostasis of these solutes, increases and decreases in sodium may also initiate, directly or indirectly, signaling cascades that regulate a variety of intracellular post-translational events. In this review, we will describe how the Na+/K+ ATPase maintains a Na+ gradient utilized by multiple sodium-dependent transport mechanisms to regulate glucose uptake, excitatory neurotransmitters, calcium signaling, acid-base balance, salt-wasting disorders, fluid volume, and magnesium transport. We will discuss how several Na+-dependent cotransporters and Na+-dependent exchangers have significant roles in human health and disease. Finally, we will discuss how each of these Na+-dependent transport mechanisms have either been shown or have the potential to use Na+ in a secondary role as a signaling molecule.

Loss of the Na+/H+ Exchange Regulatory Factor 1 Increases Susceptibility to Cisplatin-Induced Acute Kidney Injury.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase-associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.

Water Homeostasis and Cell Volume Maintenance and Regulation.

From early unicellular organisms that formed in salty water environments to complex organisms that live on land away from water, cells have had to protect a homeostatic internal environment favorable to the biochemical reactions necessary for life. In this chapter, we will outline what steps were necessary to conserve the water within our cells and how mechanisms have evolved to maintain and regulate our cellular and organismal volume. We will first examine whole body water homeostasis and the relationship between kidney function, regulation of blood pressure, and blood filtration in the process of producing urine. We will then discuss how the composition of the lipid-rich bilayer affects its permeability to water and salts, and how the cell uses this differential to drive physiological and biochemical cellular functions. The capacity to maintain cell volume is vital to epithelial transport, neurotransmission, cell cycle, apoptosis, and cell migration. Finally, we will wrap up the chapter by discussing in some detail specific channels, cotransporters, and exchangers that have evolved to facilitate the movement of cations and anions otherwise unable to cross the lipid-rich bilayer and that are involved in maintaining or regulating cell volume.

Na+ -K+ -2Cl- Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia.

Two genes encode the Na+ -K+ -2Cl- cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na+ , 1K+ , and 2Cl- across the plasma membrane of cells. Na+ -K+ -2Cl- cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na+ /K+ pump. In many cells, NKCC1 accumulates Cl- above its electrochemical potential equilibrium, thereby facilitating Cl- channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca2+ channels, leading to Ca2+ influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl- conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl- . In combination with the Na+ /K+ pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl- -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K+ -driven fluid secretion in inner ear, and possibly in Na+ -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na+ /K+ pump participates in reabsorbing 30% of the glomerular-filtered Na+ . Overall, many critical physiological functions are maintained by the activity of the two Na+ -K+ -2Cl- cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.

Design of a mouse restraint for synchrotron-based computed tomography imaging.

High-resolution computed tomography (CT) imaging of a live animal within a lead-lined synchrotron light hutch presents several unique challenges. In order to confirm that the animal is under a stable plane of anaesthesia, several physiological parameters (e.g. heart rate, arterial oxygen saturation, core body temperature and respiratory rate) must be remotely monitored from outside the imaging hutch. In addition, to properly scan the thoracic region using CT, the animal needs to be held in a vertical position perpendicular to the fixed angle of the X-ray beam and free to rotate 180°-360°. A new X-ray transparent mouse restraint designed and fabricated using computer-aided design software and three-dimensional rapid prototype printing has been successfully tested at the Biomedical Imaging and Therapy bending-magnet (BMIT-BM) beamline at the Canadian Light Source.

Physiology of SLC12 transporters: lessons from inherited human genetic mutations and genetically engineered mouse knockouts.

Among the over 300 members of the solute carrier (SLC) group of integral plasma membrane transport proteins are the nine electroneutral cation-chloride cotransporters belonging to the SLC12 gene family. Seven of these transporters have been functionally described as coupling the electrically silent movement of chloride with sodium and/or potassium. Although in silico analysis has identified two additional SLC12 family members, no physiological role has been ascribed to the proteins encoded by either the SLC12A8 or the SLC12A9 genes. Evolutionary conservation of this gene family from protists to humans confirms their importance. A wealth of physiological, immunohistochemical, and biochemical studies have revealed a great deal of information regarding the importance of this gene family to human health and disease. The sequencing of the human genome has provided investigators with the capability to link several human diseases with mutations in the genes encoding these plasma membrane proteins. The availability of bacterial artificial chromosomes, recombination engineering techniques, and the mouse genome sequence has simplified the creation of targeting constructs to manipulate the expression/function of these cation-chloride cotransporters in the mouse in an attempt to recapitulate some of these human pathologies. This review will summarize the three human disorders that have been linked to the mutation/dysfunction of the Na-Cl, Na-K-2Cl, and K-Cl cotransporters (i.e., Bartter's, Gitleman's, and Andermann's syndromes), examine some additional pathologies arising from genetically modified mouse models of these cotransporters including deafness, blood pressure, hyperexcitability, and epithelial transport deficit phenotypes.

A molecular analysis of the Na(+)-independent cation chloride cotransporters.

The homologous genes encoding the electroneutral solute carrier family 12A (SLC12A) were identified more than 20 years ago, however, over the last few years, it has become clear that each of the genes within this family potentially encode for more than one cation-chloride cotransporter (CCC). Even more surprising, despite more than 30 years of functional studies and a wealth of knowledge on the activators, inhibitors, ion affinities, and kinetics of these cotransporters, we still cannot sufficiently explain why some cells express only one CCC isoform, while others express two, three, or more CCC isoforms. In 2009, Drs. Alvarez-Leefmans and Di Fulvio published an extensive in silico molecular analysis of the potential splice variants of the Na(+)-dependent cation-chloride cotransporters. In this review, we will look at the exceptionally large variety of potential splice variants within the Na(+)-independent cation-chloride cotransporter (SLC12A4-SLC12A7) genes, their initial tissue identification, and their physiological relevance.

Calcium-binding protein 39 facilitates molecular interaction between Ste20p proline alanine-rich kinase and oxidative stress response 1 monomers.

X-ray crystallography of the catalytic domain of oxidative stress response 1 (OSR1) has provided evidence for dimerization and domain swapping. However, the functional significance of dimer formation or domain swapping has yet to be addressed. In this study, we used nine glutamine residues to link the carboxyl end of one SPAK (related Ste20 kinase) monomer to the amino end of another SPAK monomer to assess the role of kinase monomers versus dimers in Na-K-2Cl cotransporter 1 (NKCC1) activation. Transport studies in Xenopus laevis oocytes show that forcing dimerization of two wild-type SPAK molecules results in cotransporter activation when calcium-binding protein 39 (Cab39) is coexpressed, indicating that the presence of Cab39 can bypass the upstream phosphorylation requirement of SPAK normally associated with kinase activation. We determined that monomers are the functional units of the kinase as concatamers consisting of an active and various inactive monomers were still functional. Furthermore, we found that two different nonfunctional SPAK mutants could be linked together in a concatamer and activated, presumably by domain swapping, indicating that dimerization and domain swapping are both important components of kinase activation. Finally, we demonstrate rescue of a nonfunctional SPAK mutant by domain swapping with wild-type OSR1, indicating that heterodimers of the two Ste20-related kinases are possible and therefore potentially relevant to the regulation of NKCC1 activity.

Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport.

SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI subfamily of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant, and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration.

High-grade glioma motility reduced by genetic knockdown of KCC3.

Cell motility is dependent on a coordinated reorganization of the cytoskeleton, membrane recycling, and focal adhesion to the extracellular matrix. Each of these cellular processes involves re-distribution of cell water, which is facilitated by the transport of inorganic ions (with obligatory water movement). Scratch-wound healing assays of Wistar C6 glioblastoma cells demonstrated cell motility in advance of cell proliferation. Although bumetanide inhibition of Na-K-2Cl cotransport activity did not affect cell motility, treatment of glioma cells with furosemide to inhibit K-Cl cotransport activity prevented ~75% of wound closure in a reversible reaction. Genetic silencing of KCC3 with short hairpin interfering RNA reduced protein expression by 40 - 60%, K(+) influx by ~50%, and cell motility by ~50%. Appearance of KCC1 mRNA and KCC3 mRNA at 25 PCR cycles versus KCC4 mRNA at 35 PCR cycles, suggests more KCC1/KCC3 expression in both primary rat astrocytes and C6 glioma cells. Altogether, these experiments suggest that the presence/function of multiple isoforms of the Na(+-)independent K-Cl cotransporter may have a role in glioma cell motility.

Functional insights into the activation mechanism of Ste20-related kinases.

Mammalian Ste20-related kinases modulate salt transport and ion homeostasis through physical interaction and phosphorylation of cation-chloride cotransporters. Identification of a sea urchin (Strongylocentrotus purpuratus) ortholog of the mouse Oxidative Stress Response 1 (OSR1) kinase prompted the cloning and testing of the functional effect of a non-mammalian kinase on a mammalian cotransporter. Heterologous expression of sea urchin OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevisoocytes activated the cotransporter indicating evolutionary conservation of the WNK4-OSR1-NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full-length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras by replacing specific portions of the suOSR1 gene with complimentary mouse OSR1 sequences. Co-expression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which possibly prevented the kinase from acquiring an active conformation. Interestingly, non-functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co-expressed in frog oocytes. Sea urchin/mouse OSR1 chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20-related kinases.

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes.

A detailed study of hypertonically stimulated Na-K-2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to better understand the 1 K(+):1 Cl(-) stoichiometry of transport that was previously observed. In this study, we derived the velocity equations for K(+) influx under both rapid equilibrium assumptions and combined equilibrium and steady-state assumptions and demonstrate that the behavior of the equations and curves in Lineweaver-Burke plots are consistent with a model where Cl(-) binds first, followed by Na(+), a second Cl(-), and then K(+). We further demonstrate that stimulation of K(+) movement by K(+) on the trans side is an intrinsic property of a carrier that transports multiple substrates. We also demonstrate that K(+) movement through NKCC1 is strictly dependent upon the presence of external Na(+), even though only a fraction of Na(+) is in fact transported. Finally, we propose that the larger transport of K(+), as compared with Na(+), is a result of the return of partially unloaded carriers, which masks the net 1Na(+):1K(+):2Cl(-) stoichiometry of NKCC1. These data have profound implications for the physiology of Na-K-2Cl cotransport, since transport of K-Cl in some conditions seems to be uncoupled from the transport of Na-Cl.

Housing and husbandry of Xenopus laevis affect the quality of oocytes for heterologous expression studies.

To assess the effect of Xenopus husbandry on oocyte quality for membrane transport physiology experiments, we compared a recirculating-water housing system with a static-water system in a 23-mo study. Two groups of frogs (n = 8) were maintained separately for the entire study: one group was housed in a multiinvestigator centrally managed Xenopus facility, which consists of 33 tanks placed on a shared and recirculating water system; the other group was housed in a satellite facility used by a single investigator and consisting of static tanks placed in a dedicated cold-room. The activity of a heterologously expressed membrane transporter was assessed every 4 to 5 wk for a total of 23 mo. Activity of the mouse cotransporter NKCC1 was assessed through isotopic (86) Rb influx measurements under 2 experimental conditions: stimulation of cotransporter by coinjection of regulatory kinases and by exposure to a hypertonic solution. The results showed a significant difference in the level of ion fluxes under these 2 experimental conditions between the 2 groups of oocytes. During the entire period, oocytes isolated from frogs maintained in the static facility demonstrated consistently robust NKCC1 function, whereas oocytes isolated from frogs maintained in the recirculating facility showed inconsistent and weaker cotransporter function. Furthermore, the oocytes isolated from frogs maintained in the recirculating facility showed significant deterioration during the summer months (April to August), a seasonal variation that was muted in frog oocytes maintained in the static facility.

Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchange.

Na(+)-K(+)-2Cl(-) cotransport (NKCC) mediates the movement of two Cl(-) ions for one Na(+) and one K(+) ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl(-) uptake in Xenopus laevis oocytes, as measured using (36)Cl, is twice the value of K(+) uptake, as determined using (86)Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K(+) uptake with only a minimal increase in bumetanide-sensitive Cl(-) uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K(+)/K(+) and Cl(-)/Cl(-) exchange. Although significant stimulation of K(+)/K(+) exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K(+)/K(+) exchange.