82Se Metabolically-Labeled Yeast as a Matrix-Matched Isotope Dilution Standard for Quantification of Selenomethionine.

Selenized yeast is commonly used as a highly bioavailable source of selenium in dietary supplements and feed additives and is used in research settings in various disciplines due to the large number of selenium-containing metabolites formed during growth. With the selenomethionine being the major form of selenium present in selenized yeasts, its accurate quantitation is essential, however, values are frequently underestimated due to the costly and time-consuming hydrolysis-based sample preparation required to release the selenoamino acid from proteins for analysis. The National Research Council Canada has developed an 82-Se-enriched selenized yeast Certified Reference Material, SEEY-1 (DOI: 10.4224/crm.2023.seey-1) intended to be used as a matrix-matched spike material for isotope dilution analysis of selenized yeasts. The total selenium and selenomethionine contents of SEEY-1 were determined to be 322.1 ± 4.8 mg/kg (k = 2) and 635.6 ± 16.8 mg/kg (k = 2), respectively. Here we present results on the preparation of the 82-Se-enriched yeast, the certification process, and provide an example of the use of SEEY-1 as a matrix-matched spike for the analysis of selenomethionine in a sample of selenized yeast. We demonstrate here that SEEY-1 is able to compensate for the partial digestion of yeast proteins and provide reliable analytical data on Se amino acid content in under an hour instead of the 16 hours required for conventional complete acid hydrolysis.

Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Chloride extrusion enhancers as novel therapeutics for neurological diseases.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Distribution, burden, and impact of acute gastroenteritis in Dominica, 2009-2010.

Acute gastroenteritis (AGE) is an important public-health issue in Dominica. To determine the burden of AGE in Dominica, a retrospective, cross-sectional population survey was conducted in March-April 2009 and October 2010 (low- and-high-AGE seasons) and a laboratory survey from April 2009 to March 2010. The overall monthly prevalence of self-reported AGE was 8.6 % (95% CI 7.0-10.6); the incidence rate was 1.1 episodes/person-year and 79,157.1 episodes of AGE for the total population/year. Monthly prevalence of AGE was the highest in the 1-4 year(s) age-group (25.0%), higher in females (10.8%) and also varied by health district, with the highest monthly prevalence of AGE being reported in the Portsmouth district (13.1%). This difference in gender and across the health region was statistically significant. The estimated underreporting of syndromic AGE to the Ministry of Health was 83.3%. Furthermore, for every reported laboratory-confirmed case of AGE and foodbome disease (FBD), there was an estimated underreporting factor of 280. Overall, 47% of AGE specimens tested were positive for FBD pathogens. The predominant pathogens isolated were norovirus, followed by Giardia, Salmonella, and Shigella. The total annual estimated cost of AGE was US$ 1,371,852.92, and the total cost per capita due to AGE was US$ 19.06, indicating an economic burden of AGE-related illness on a small island of Dominica.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B.

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Heavy metal cations permeate the TRPV6 epithelial cation channel.

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Phosphoregulation of K(+)-Cl(-) cotransporter 4 during changes in intracellular Cl(-) and cell volume.

It has long been stated that the K(+)-Cl(-) cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N-ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C(VOL)) and intracellular [Cl(-)] ([Cl(-)](i)) on the activity and phosphorylation levels (P(LEV)) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)-sensitive effectors. We found that (1) low [Cl(-)](i) or low C(VOL) leads to decreased activity but increased P(LEV), (2) high C(VOL) leads to increased activity but no decrease in P(LEV) and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P(LEV). Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl(-)](i) or C(VOL) modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A-sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787-796, 2009. (c) 2009 Wiley-Liss, Inc.

Modulation of gene expression in human macrophages treated with the anti-leishmania pentavalent antimonial drug sodium stibogluconate.

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan parasite that resides and multiplies exclusively in the phagolysosomes of macrophages. Leishmania control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam) or meglumine antimoniate (Glucantime). The mode of action of SbV is still not known precisely. To explore the effect of SbV on macrophage gene expression, a microarray analysis was performed using Affymetrix focus arrays to compare gene expression profiles in noninfected and L. donovani-infected THP-1 monocytic cells treated or not treated with sodium stibogluconate. Under our experimental conditions, SbV changed the expression of a few host genes, and this was independent of whether cells were infected or not infected with Leishmania. Leishmania infection had a greater effect on the modulation of host gene expression. Statistical analyses have indicated that the expression of eight genes was modified by at least twofold upon SbV treatment, with six genes upregulated and two genes downregulated. One gene whose expression was affected by SbV was the heme oxygenase gene HMOX-1, and this change was observed both in the monocytic cell line THP-1 and in primary human monocyte-derived macrophages. Another pathway that was affected was the glutathione biosynthesis pathway, where the expression of the glutamate-cysteine ligase modifier subunit was increased upon SbV treatment. Our analysis has suggested that, under our experimental conditions, the expression of a few genes is altered upon SbV treatment, and some of these encoded proteins may be implicated in the yet-to-be-defined mode of action of SbV.

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.

Trypanosoma cruzi-mediated IFN-gamma-inducible nitric oxide output in macrophages is regulated by iNOS mRNA stability.

Although the effects of activated macrophages (Muphi) on the intracellular parasite Trypanosoma cruzi are well documented, little is known about how host-Muphi functions are affected by this pathogen before activation. This study is aimed at assessing the capacity of T. cruzi infection to modulate J77.4 murine Muphi NO generation following IFN-gamma stimulation, and identifying mechanisms regulating this modulation. Results show that parasite infection potentiates Muphi to produce inducible NO synthase (iNOS) mRNA and protein as well as NO following IFN-gamma stimulation above IFN-gamma alone controls. This potentiation occurs through the concomitant activation of NF-kappaB, ERK1/ERK2 MAPK, and stress-activated protein kinase signaling pathways. Activation of the JAK/STAT pathway by IFN-gamma then leads to STAT1alpha translocation and the transcription of a stable iNOS mRNA species. A decreased rate of iNOS mRNA degradation results in elevated levels of iNOS protein and NO production. Maximal iNOS expression is likely achieved through NF-kappaB activation by T. cruzi, whereas iNOS mRNA stability results from ERK1/ERK2 MAPK and stress-activated protein kinase activation by the infection. Taken together, our data show that T. cruzi-infected Muphi NO generation is controlled at both pre- and posttranscriptional levels and relies on signaling pathway cross-talk. This is the first report of a parasite pathogen capable of heightening host mRNA stability.

Identification of key functional domains in the C terminus of the K+-Cl- cotransporters.

The K+-Cl- cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4beta-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.

Statins could be used to control replication of some viruses, including HIV-1.

Statins are mainly known for their plasma cholesterol-lowering properties and are widely used for the prevention of cardiovascular diseases. They however also exert pleiotropic effects through a variety of mechanisms, among which several immunosuppressive effects that are unrelated to their cholesterol-lowering activity. Interestingly, there has been recent evidence of antiviral effects, including preliminary studies on the efficacy of statins against HIV-1. This paper more particularly focuses on the specific inhibition of the binding of leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule (ICAM-1) by statins, independently of the inhibition of HMGCoA reductase. Targeting the statin-binding site within LFA-1 or regulating LFA-1 affinity by inhibiting prenylation of the small GTPases could prove useful to treat inflammatory, autoimmune diseases and possibly viral infections, including HIV-1.

Novel insights regarding the operational characteristics and teleological purpose of the renal Na+-K+-Cl2 cotransporter (NKCC2s) splice variants.

The absorptive Na(+)-K(+)-Cl(-) cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H(2)O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H(2)O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2-NKCC2 interactions.

Molecular mechanisms of cation transport by the renal Na+-K+-Cl- cotransporter: structural insight into the operating characteristics of the ion transport sites.

Two variants of the renal Na(+)-K(+)-Cl(-) cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na(+), Rb(+), and Cl(-) affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an alpha-helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A-F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structure-function analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K(m (C1-)), suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K(m (Rb+)) while the other (residue 220) only affected K(m (Na+)). We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 alpha-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.

Influenza virus activates human immunodeficiency virus type-1 gene expression in human CD4-expressing T cells through an NF-kappaB-dependent pathway.

Influenza virus infection can cause severe complications in human immunodeficiency virus type-1 (HIV-1)-infected individuals leading to an increased risk of complications and death compared to that seen in uninfected individuals. We assessed the capacity of influenza virus (Flu) to modulate transcription of the HIV-1 long terminal repeat (LTR) in human CD4+ T cells. We found that Flu is able to promote expression of both the transiently transfected and stably integrated HIV-1 LTR-driven reporter gene. Experiments performed with Arthrobacter-derived neuraminidase and ammonium chloride revealed that Flu-dependent activation of HIV-1 transcription required an intimate contact between Flu and the target cell and efficient entry of Flu inside human CD4+ T cells. Amplification of a Flu-specific mRNA by RT-PCR indicated that human T cells were indeed productively infected with Flu. Virus preparations rendered noninfectious after UV irradiation could no longer upregulate HIV-1 LTR activity. Furthermore, experiments conducted with wild type and NF-kappaB-mutated HIV-1 LTR-directed reporter vectors suggested that the positive action of Flu on HIV-1 LTR activity was mediated through the induction of NF-kappaB. Our data show that fully competent Flu can lead to NF-kappaB-dependent activation of HIV-1 transcription in CD4+ T cells.

Characterization of a novel interaction between the secretory Na+-K+-Cl- cotransporter and the chaperone hsp90.

The first isoform of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl(-)] (Cl(-)(i)) represents a key signaling mechanism by which NKCC1-induced Cl(-) movement is autoregulated and by which Cl(-) entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl(-)(i)-dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na(+)-K(+)-Cl(-) cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction.

Molecular mechanisms of Cl- transport by the renal Na(+)-K(+)-Cl- cotransporter. Identification of an intracellular locus that may form part of a high affinity Cl(-)-binding site.

The 2nd transmembrane domain (tm) of the secretory Na(+)-K(+)-Cl(-) cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were approximately 8 mm for saF/A and saA, and approximately 70 mm for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all approximately 20 mM, and Km(Rb+) all approximately 2 mM. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.