Molecular motions involved in Na-K-Cl cotransporter-mediated ion transport and transporter activation revealed by internal cross-linking between transmembrane domains 10 and 11/12.

We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.

Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1).

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.

MAL/VIP17, a new player in the regulation of NKCC2 in the kidney.

The renal-specific Na+-K+-2Cl- cotransporter (NKCC2) is the major salt transport pathway of the apical membrane of the mammalian thick ascending limb of Henle's loop. Here, we analyze the role of the tetraspan protein myelin and lymphocytes-associated protein (MAL)/VIP17 in the regulation of NKCC2. We demonstrated that 1) NKCC2 and MAL/VIP17 colocalize and coimmunoprecipitate in Lilly Laboratories cell porcine kidney cells (LLC-PK1) as well as in rat kidney medullae, 2) a 150-amino acid stretch of NKCC2 C-terminal tail is involved in the interaction with MAL/VIP17, 3) MAL/VIP17 increases the cell surface retention of NKCC2 by attenuating its internalization, and 4) this coincides with an increase in cotransporter phosphorylation. Interestingly, overexpression of MAL/VIP17 in the kidney of transgenic mice results in cysts formation in distal nephron structures consistent with the hypothesis that MAL/VIP17 plays an important role in apical sorting or in maintaining the stability of the apical membrane. The NKCC2 expressed in these mice was highly glycosylated and phosphorylated, suggesting that MAL/VIP17 also is involved in the stabilization of NKCC2 at the apical membrane in vivo. Thus, the involvement of MAL/VIP17 in the activation and surface expression of NKCC2 could play an important role in the regulated absorption of Na+ and Cl- in the kidney.

Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells.

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the "secretory" NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells.

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na(+) reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na(+) transport. Previous studies have shown that SGK1 increases Na(+) transport and epithelial Na(+) channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na(+)-K(+)-ATPase activity, the transporter responsible for basolateral Na(+) efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na(+)-K(+)-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1(T)(S425D)) increased the transport activity of Na(+)-K(+)-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na(+)-K(+)-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na(+) pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1(T)(S425D), induced an approximately 2.5-fold increase in total protein and plasma membrane Na(+)-K(+)-ATPase alpha(1)-subunit abundance. We conclude that aldosterone increases the abundance of Na(+)-K(+)-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli.

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter.

Brain edema that forms during the early stages of stroke involves increased transport of Na+ and Cl- across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na+-K+-Cl- cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na+-K+-Cl- cotransporter, as well as on Na+-K+-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na+-K+-Cl- cotransporter and Na+-K+-ATPase activities as bumetanide-sensitive and ouabain-sensitive 86Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [3H]-3-O-methyl-D-glucose and [14C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O2 (vs. 19% normoxic O2) significantly increased cotransporter activity as did 7.5% or 2% O2 for up to 2 h. This was not associated with reduction in Na+-K+-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na+-K+-Cl- cotransporter contributes to ischemia-induced brain edema formation.

Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer.

Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na(+)-K(+)-ATPase-alpha(1a) (3-fold), Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na(+)-K(+)-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na(+)-K(+)-ATPase-alpha(1a) mRNA expression and Na(+)-K(+)-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H(+)-ATPase expression, supporting the current suggestion that killifish gills transport Na(+) via Na(+)/H(+) exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.

Functional comparison of renal Na-K-Cl cotransporters between distant species.

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm(2)). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm(2) in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of (86)Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm(2) of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.