Expression of the Na+-K+-2Cl- cotransporter in the rat endolymphatic sac

The endolymphatic sac (ES) is a part of the membranous labyrinth that contains the cochlea, vestibular organs, and semicircular canals, and is believed to absorb endolymphatic fluid. Na⁺–K⁺–2Cl⁻ (NKCC) is a cotransporter that occurs as two isoforms (NKCC-1 and NKCC-2). Especially, NKCC-2 is suggested to participate in ES endolymph absorption. In the present study, the expression and cellular localization of NKCC-1 and NKCC-2 in the rat ES were examined by RT-PCR and in situ hybridization, respectively. The findings indicate that both NKCC-1 and NKCC-2 are expressed in the rat ES and suggest that NKCC is involved in ES homeostasis. NKCC-2 may be particularly involved in endolymph absorption. This is the first report confirming NKCC expression in the ES.     

Purinergic stimulation induces Ca2+-dependent activation of Na+-K+-2Cl- cotransporter in human nasal epithelia

Increasing evidence suggests that P2 receptors (P2Rs) in airway epithelial cells perform critical functions in auto- or paracrine regulation of fluid and mucus secretion. In the present study, we characterized the effects of P2R stimulation on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in normal human nasal epithelial (NHNE) cells. [Ca(2+)](i) and pH(i) were measured in primary cultures of NHNE cells using a double perfusion chamber, which enabled us to analyze membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to Na(+)-dependent and bumetanide-sensitive intracellular uptake of NH(4)(+). NKCC activities were observed in the basolateral membrane, but not in the luminal membrane, of NHNE cells. Interestingly, P2Rs were expressed in both membranes, and the stimulation of either luminal or basolateral P2R increased NKCC activity. Blockades of luminal Cl(-) channels, basolateral K(+) channels, or protein kinase C did not affect the activation of NKCC by basolateral P2R stimulation. The effects of luminal P2R stimulation were partially reduced by Cl(-) channel blockers. However, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) treatment completely blocked the stimulatory effects of luminal and basolateral P2Rs on NKCC. In addition, increasing [Ca(2+)](i) by treatment with ionomycin-stimulated NKCC activity. These results provide evidence that stimulation of P2Rs directly activates basolateral NKCC by Ca(2+)-dependent pathways in NHNE cells, which is an important aspect of the purinergic regulation of ion and fluid secretions in human airway epithelia under physiologic and pathologic conditions.     

Transmembrane topology of the secretory Na+-K+-2Cl- cotransporter NKCC1 studied by in vitro translation

The secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is a member of a small gene family of electroneutral salt transporters. Hydropathy analyses indicate that all of these transporters have a similar general structure consisting of large hydrophilic N and C termini on either side of a central, relatively well conserved, hydrophobic domain. Programs that predict the transmembrane topology of polytopic membrane proteins identify 10-12 putative membrane-spanning segments (MSSs) in this hydrophobic domain; but to date, there is little experimental data on the structure of this region for any of these transporters. In this report, we have studied the transmembrane topology of NKCC1 using an in vitro translation system designed to test the membrane insertion properties of putative MSSs (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Fusion proteins consisting of putative NKCC1 MSSs inserted either (i) between an N-terminal cytosolic anchor sequence and a C-terminal reporter sequence containing multiple N-linked glycosidation sites or (ii) between an N-terminal signal anchor sequence and the same glycosidation flag were expressed in the presence of canine pancreatic microsomes. The glycosidation status of the reporter sequence, which indicated its luminal or extraluminal location in the microsomes, was then used to characterize the signal anchor or stop transfer activity of the inserted MSSs. The results of this experimental analysis yielded a topology scheme consisting of 12 membrane-spanning segments, two pairs of which apparently form rather tight hairpin-like structures within the membrane.     

Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1)

The secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 microm), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.     

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.     

Evidence that up-regulation of the parotid Na+-K+-2Cl- cotransporter by hypertonic shrinkage is not duplicated by isotonic shrinkage

The application of Ca2+ mobilizing secretagogues to rat parotid acini results in a significant decrease in cell volume (15-30%) due to isotonic salt loss. It is often assumed that the effects of such an isotonic volume decrease can be mimicked by anisotonic cell shrinkage. We demonstrate that the Na+-K+-2Cl- cotransporter in these cells is up-regulated by Ca2+ mobilizing secretagogues as well as by cell shrinkage in hypertonic media. However, we find that although the protein kinase inhibitors staurosporine (0.3 M) and K252a (0.6 M) significantly blunt the latter up-regulation, they are without effect on the former. These observations suggest that hypertonic and isotonic shrinkage do not result in the activation of the same intracellular signalling pathways, and indicate that anisotonic volume perturbations may not provide good experimental models of physiologic isotonic volume changes.     

Biogenesis and topology of the secretory Na+-K+-2Cl- cotransporter (NKCC1) studied in intact mammalian cells

The "secretory" Na+-K+-2Cl- cotransporter (NKCC1) is a member of a small gene family with nine homologues in vertebrates. Of these, seven are known to be electroneutral chloride transporters. These transporters play a number of important physiological roles related to salt and water homeostasis and the control of intracellular chloride levels. Hydropathy analyses suggest that all of these transporters have a similar transmembrane topology consisting of relatively large intracellular N and C termini and a central hydrophobic domain containing 12 membrane-spanning segments (MSSs). In recent experiments from our laboratory [Gerelsaikhan, T., and Turner, R. J. (2000) J. Biol. Chem. 275, 40471-40477], we employed an in vitro translation system to confirm that each of the putative MSSs of NKCC1 was capable of membrane integration in a manner consistent with a 12 MSS model. Here, we extend that work to the study of the biogenesis of NKCC1 in intact cells. We employ a truncation mutant approach that allows us to monitor this process quantitatively as successive MSSs are synthesized. While the results presented here confirm the 12 MSS model, they also indicate that the integration of NKCC1 into the membrane does not occur via a simple cotranslational process. In particular, we demonstrate that two MSSs, the second and sixth, require the presence of downstream sequence to efficiently integrate into the membrane.     

Tumor necrosis factor-alpha is an endogenous inhibitor of Na+-K+-2Cl- cotransporter (NKCC2) isoform A in the thick ascending limb

The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ～40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ～60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ～30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.     

Regulation of Na(+)-K(+)-2Cl- cotransporter activity in rat skeletal muscle and intestinal epithelial cells

In mammalian cells, Na(+)-K(+)-2Cl- cotransporter activity participates in regulation of ion and volume homeostasis. This makes NKCC indispensable for normal cell growth and proliferation. We recently reported the existence of two mechanisms that can regulate NKCC activity in mature skeletal muscle. In isosmotic conditions, signaling through ERK MAPK pathway is necessary, while inhibition of the cAMP-dependent protein kinase A (PKA) pathway stimulates NKCC activity during hyperosmotic challenge. Both pathways are involved in regulating cell proliferation in wide variety of cells of epithelial and non-epithelial origin, so we tested which pathway regulated NKCC activity in cultured cells. In cultured rat skeletal muscle (L6) and intestinal epithelial (IEC-6) cells, NKCC activity in the basal state comprised 30 to 50% of total potassium influx, assessed as bumetanide-sensitive 38Rb-uptake. This NKCC activity could not be abolished by inhibitors of ERK MAPK (PD98059 and U0126), PKC (GF109203X), or PI 3-K (wortmannin, LY294002). In L6 myoblasts and in IEC-6 cells, elevation of cAMP levels with isoproterenol or forskolin led to a 60% inhibition on NKCC activity. In contrast, incubation of IEC-6 cells with the PKA-inhibitor H-89 resulted in 50% increase of NKCC activity compared with the basal level. In conclusion, it appears that in cultured cells the cAMP--PKA pathway regulates NKCC activity. This resembles hyperosmotic regulation of NKCC activity.     

Role of Cl- in cerebral vascular tone and expression of Na+-K+-2Cl- co-transporter after neonatal hypoxia-ischemia

Chloride (Cl-) efflux induces depolarization and contraction of vascular smooth muscle cells. In the basilar arteries from the New Zealand white rabbits, the role of Cl- flux in serotonin-induced contraction was demonstrated by (i) inhibition of Na+-K+-2Cl- co-transporter (NKCC1) to decreased Cl- influx with bumetanide; (ii) a disabled Cl-/HCO3- exchanger with bicarbonate free HEPES solution; (iii) blockade of Cl- channels using 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94, R-(+)-methylindazone (R-(+)-IAA-94); and (iv) substitution of extracellular Cl- with methanesulfonate acid (113 mmol/L; Cl-, 10 mmol/L). In addition, the expression of NKCC1 in brain tissues after neonatal hypoxia-ischemia was examined at mRNA and protein levels using RT-PCR and Western blotting techniques. NKCC1 mRNA and protein expressions were increased at 24 and 48 h and returned to normal levels at 72 h after hypoxia insult when compared with the control littermates. In conclusion, Cl- efflux regulates cerebral circulation and the up-regulation of NKCC1 after neonatal hypoxia-ischemia may contribute to brain injury.     

Impaired cardiac contractility in mice lacking both the AE3 Cl-/HCO3- exchanger and the NKCC1 Na+-K+-2Cl- cotransporter: effects on Ca2+ handling and protein phosphatases

To analyze the cardiac functions of AE3, we disrupted its gene (Slc4a3) in mice. Cl(-)/HCO3(-) exchange coupled with Na+-dependent acid extrusion can mediate pH-neutral Na+ uptake, potentially affecting Ca2+ handling via effects on Na+/Ca2+ exchange. AE3 null mice appeared normal, however, and AE3 ablation had no effect on ischemia-reperfusion injury in isolated hearts or cardiac performance in vivo. The NKCC1 Na+-K+-2Cl(-) cotransporter also mediates Na+ uptake, and loss of NKCC1 alone does not impair contractility. To further stress the AE3-deficient myocardium, we combined the AE3 and NKCC1 knock-outs. Double knock-outs had impaired contraction and relaxation both in vivo and in isolated ventricular myocytes. Ca2+ transients revealed an apparent increase in Ca2+ clearance in double null cells. This was unlikely to result from increased Ca2+ sequestration, since the ratio of phosphorylated phospholamban to total phospholamban was sharply reduced in all three mutant hearts. Instead, Na+/Ca2+ exchanger activity was found to be enhanced in double null cells. Systolic Ca2+ was unaltered, however, suggesting more direct effects on the contractile apparatus of double null myocytes. Expression of the catalytic subunit of protein phosphatase 1 was increased in all mutant hearts. There was also a dramatic reversal, between single null and double null hearts, in the carboxymethylation and localization to the myofibrillar fraction, of the catalytic subunit of protein phosphatase 2A, which corresponded to the loss of normal contractility in double null hearts. These data show that AE3 and NKCC1 affect Ca2+ handling, PLN regulation, and expression and localization of major cardiac phosphatases and that their combined loss impairs cardiac function.     

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.     

Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.

The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.     

Regulation of the renal-specific Na+-K+-2Cl- co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.     

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.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.