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Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K + channel. However, the expression of CFTR in apical cell membranes or its function as a Cl − channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl − channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl − channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl − channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl − channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR inh -172 abolished apical Cl − channel activity in excised patches. Moreover, apical Cl − channel activity was completely absent in principal cells from transgenic mice expressing the ΔF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl − channels on salt handling by the collecting duct and on the functional CFTR–ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

Digestion of food and normal salt and water homeostasis in the body require a functional digestive tract. Recently an increasing number of studies have demonstrated a role for the calcium-sensing receptor along the entire gastrointestinal tract and its role in normal gut physiology. Detailed studies have been performed on colonic fluid transport and gastric acid secretion. We have now demonstrated that the receptor can modulate fluid secretion and absorption along the intestine and can thereby be a potent target to prevent secretory diarrhea. Recent studies have demonstrated that organic nutrients such as polyamines and l-amino acids can act as agonists by allosterically modifying the receptor. Thus, the receptor may detect nutrient availability to epithelial cells along the gastrointestinal tract and may be involved in the coordinated rapid turnover of the intestinal epithelium. Furthermore, the receptor has been suggested as a link for the mechanisms leading to calcium uptake by the colon and may thus reduce the risk for colon cancer.

ROMK null mice with a high survival rate and varying severity of hydronephrosis provide a good model to study type II Bartter syndrome pathophysiology ( 26 ). During the development of such a colony, we found that more male than female null mice survived, 58.7% vs. 33.3%. To investigate the possible mechanism of this difference, we compared the survival rates, renal functions, degree of hydronephrosis, as well as PGE2 and TXB2 production between male and female ROMK wild-type and null mice. We observed that female ROMK Bartter's mice exhibited lower GFR (0.37 vs. 0.54 ml·min−1·100 g BW−1, P < 0.05) and higher fractional Na+ excretion (0.66% vs. 0.48%, P < 0.05) than male Bartter's. No significant differences in acid-base parameters, urinary K+ excretion, and plasma electrolyte concentrations were observed between sexes. In addition, we assessed the liquid retention rate in the kidney to evaluate the extent of hydronephrosis and observed that 67% of male and 90% of female ROMK null mice were hydronephrotic mice. Urinary PGE2 excretion was higher in both sexes of ROMK null mice: 1.35 vs. 1.10 ng/24 h in males and 2.90 vs. 0.87 ng/24 h in females. TXB2 excretion was higher in female mice in both wild-type and ROMK null mice. The increments of urinary PGE2 and TXB2 were significantly higher in female null mice than males, 233.33% vs. 22.74% of PGE2 and 85.67% vs. 20.36% of TXB2. These data demonstrate a more severe Bartter phenotype in female ROMK null mice, and higher PGE2 and TXB2 production may be one of the mechanisms of this manifestation.

The Na + :K + :2Cl − cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl − ] i . The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.

Ongoing studies have developed strategies for identifying key bioactive compounds and chemical profiles in Echinacea with the goal of improving its human health benefits. Antiviral and antiinflammatory-antipain assays have targeted various classes of chemicals responsible for these activities. Analysis of polar fractions of E. purpurea extracts showed the presence of antiviral activity, with evidence suggesting that polyphenolic compounds other than the known HIV inhibitor, cichoric acid, may be involved. Antiinflammatory activity differed by species, with E. sanguinea having the greatest activity and E. angustifolia, E. pallida, and E. simulata having somewhat less. Fractionation and studies with pure compounds indicate that this activity is explained, at least in part, by the alkamide constituents. Ethanol extracts from Echinacea roots had potent activity as novel agonists of TRPV1, a mammalian pain receptor reported as an integrator of inflammatory pain and hyperalgesia and a prime therapeutic target for analgesic and antiinflammatory drugs. One fraction from E. purpurea ethanol extract was bioactive in this system. Interestingly, the antiinflammatory compounds identified to inhibit prostaglandin E(2) production differed from those involved in TRPV1 receptor activation.

Homeostasis of intravascular volume, Na + , Cl − , and K + is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin–angiotensin–aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K + channels, and paracellular Cl − flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na + channel (ENaC), the major mediator of aldosterone-sensitive Na + (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC β- and γ-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na + flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na + absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.

The renal-specific Na+-K+-2Cl−cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl−cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by86Rb+influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl−affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

The serine–threonine kinase WNK3 modulates Cl− transport into and out of cells through its regulation of SLC12A cation–Cl− cotransporters, implicating it as (one of) the long-sought Cl−/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na+–Cl− cotransporter (NCC), paracellular Cl− flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring hypertension and hyperkalaemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments – the epithelial sodium channel (ENaC), paracellular Cl− flux, and ROMK1 – using established model systems. WNK3 did not alter paracellular Cl− flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (P < 0.0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when coexpressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus – a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild-type kinase (P < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge–charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N–C- and C–C-terminal subunit–subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C–C-terminal arginine–glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular "migration machinery." We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.

Calcium and salinity sensing by the thick ascending limb: A journey from mammals to fish and back again. The roles of the CaSR in endocrine, epithelial, CNS, and other cells have been reviewed previously 17-20,27-33 . This brief review focuses on the roles of the CaSR in the thick ascending limb of Henle (TAL), and is written in honor of my mentor and long-term friend and colleague, Thomas E. Andreoli, on the occasion of his retirement. My early studies of TAL function with Tom Andreoli were the inspiration for this work.

We previously demonstrated that dietary K intake regulates the expression of Src family PTK, which plays an important role in controlling the expression of ROMK1 in plasma membrane (Wei Y, Bloom P, Lin D-H, Gu RM, and Wang WH. Am J Physiol Renal Physiol 281: F206–F212, 2001). In the present study, we used the immunofluorescence staining technique to demonstrate the presence of c-Src, a member of Src family PTK, in the thick ascending limb (TAL) and collecting duct. Confocal microscopy shows that c-Src is highly expressed in the renal cortex and outer medulla. Moreover, c-Src and ROMK are coexpressed in the same nephron segment. Also, the positive staining of c-Src is visible in tubules stained with Tamm-Horsfall glycoprotein or aquaporin-2. This suggests that c-Src is present in the TAL, cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). To study the role of PTK in the regulation of ROMK membrane expression in the TAL and CCD, we carried out immunocytochemical staining with ROMK antibody in the CCD or TAL from rats on either a high-K (HK) or K-deficient (KD) diet. A sharp membrane staining of ROMK can be observed in the TAL from rats on both HK and KD diets. However, a clear plasma membrane staining can be observed only in the CCD from rats on a HK diet but not from those on a KD diet. Treatment of the CCD from rats on a HK diet with phenylarsine oxide (PAO) decreases the positive staining in the plasma/subapical membrane and increases the ROMK staining in the intracellular compartment. However, PAO treatment did not significantly alter the staining pattern of ROMK in the TAL. Moreover, the biotinylation technique has also confirmed that neither herbimycin A nor PAO has significantly changed the biotin-labeled ROMK2 in HEK293 cells transfected with ROMK2 and c-Src. We conclude that c-Src is expressed in the TAL, CCD, and OMCD and that stimulation of PTK increases the ROMK channels in the intracellular compartment but decreases them in the apical/subapical membrane in the CCD.

Background & aims: Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca 2+ (Ca 2+ o /nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca 2+ -sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca 2+ on second messenger generation by, and on function of, rat distal colonic crypts. Methods: Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP 3 and Ca 2+ accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca 2+ and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro. Results: Polyamine (spermine > spermidine > putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP 3 ) and Ca 2+ accumulation required extracellular Ca 2+ , and the EC 50 for extracellular Ca 2+ -mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC 50 for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca 2+ (Ca 2+ o ). Conclusions: The interactions of extracellular Ca 2+ and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis1. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration (refs. 2,3). Positional cloning implicated the serine-threonine kinase WNK4 in this process4; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII5) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition6. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis.

This review describes recent advances in our understanding of the genetic heterogeneity, pathophysiology and treatment of Bartter syndrome, a group of autosomal recessive disorders that are characterized by markedly reduced or absent salt transport by the thick ascending limb of Henle. Consequently, individuals with Bartter syndrome exhibit renal salt wasting and lowered blood pressure, hypokalemic metabolic alkalosis and hypercalciuria with a variable risk of renal stones.Previously, three genes (SLC12A2, the sodium-potassium-chloride co-transporter; KCNJ1, the ROMK potassium ion channel; ClC-Kb, the basolateral chloride ion channel) had been identified as causing antenatal and 'classic' Bartter syndrome. Two additional genes have now been identified. Barttin is a beta-subunit that is required for the trafficking of CLC-K (both ClC-Ka and ClC-Kb) channels to the plasma membrane in both the thick ascending limb and the marginal cells in the scala media of the inner ear that secrete potassium ion-rich endolymph. Loss-of-function mutations in barttin thus cause Bartter syndrome with sensorineural deafness. In addition, severe gain-of-function mutations in the extracellular calcium ion-sensing receptor can result in a Bartter phenotype because activation of this G protein-coupled receptor inhibits salt transport in the thick ascending limb (a furosemide-like effect).Five genes have been identified as causing Bartter syndrome (types I-V), with the unifying pathophysiology being the loss of salt transport by the thick ascending limb. Phenotypic differences in Bartter types I-V relate to the specific physiological roles of the individual genes in the kidney and other organ systems.

We recently cloned a homologue of the bovine parathyroid calcium receptor from the kidney of a spiny dogfish ( Squalus acanthias) and termed this new protein SKCaR. SKCaR senses alterations in extracellular Mg2+after its expression in human embryonic kidney cells (Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown E, Hebert S, and Harris HW. Proc Natl Acad. Sci USA 99: 9231-9236, 2002). In this report, we used light and electron microscopic immunocytochemical techniques to study the distribution of SKCaR in dogfish kidney. SKCaR antiserum bound to the apical membranes of shark kidney epithelial cells in the following tubular segments: proximal tubules (PIa and PIIb), late distal tubule, and collecting tubule/collecting duct as well as diffusely labeled cells of early distal tubule. The highly specific distribution of SKCaR in mesial tissue as well as lateral countercurrent bundles of dogfish kidney is compatible with a role for SKCaR to sense local tubular Mg2+concentrations. This highly specific distribution of SKCaR protein in dogfish kidney could possibly work in concert with the powerful Mg2+secretory system present in the PIIa segment of elasmobranch fish kidney to affect recycling of Mg2+from putative Mg2+-sensing/Mg2+-reabsorbing segments. These data provide support for the possible existence of Mg2+cycling in elasmobranch kidney in a manner analogous to that described for mammals.

Mutations in the serine-threonine kinases WNK1 and WNK4 [ w ith n o lysine ( K ) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl − dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a “mirror image” of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na–Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na + influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Na-influx reduced 50%, P < 1 × 10 −9 , surface expression reduced 75%, P < 1 × 10 −14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K(+) channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na(+) wasting, consistent with the role of this channel in apical K(+) recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K(+) wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with approximately 25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/-), and null ROMK(-/-) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (+/+ and +/-) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(+/+); about half that of the wild-type in ROMK(+/-), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(-/-) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K(+) absorption/secretion in the nephron.

The calcium-sensing receptor (CaSR) is activated by extracellular calcium (Ca[Formula: see text]) and mediates many of the known effects of extracellular divalent minerals on body cells. Both surface and crypt cells express CaSR transcripts and protein on both apical and basolateral surfaces. Raising Ca[Formula: see text] elicited increases in intracellular calcium (Ca[Formula: see text]) in both surface and crypt cells with an EC50 of 2 mM. The Ca[Formula: see text]-induced increase in Ca[Formula: see text] was associated with increases in inositol 1,4,5-trisphosphate and eliminated by U-73129, an inhibitor of phosphatidylinositol-phospholipase C, as well as by thapsigargin. Other CaSR agonists, Gd3+ and neomycin, mimicked these Ca[Formula: see text]-induced responses. Both luminal and bath Ca[Formula: see text], Gd3+, and neomycin induced increases in Ca[Formula: see text] in isolated perfused crypts. The stimulatory effect of forskolin on net fluid secretion in perfused crypts was abolished by increasing Ca[Formula: see text] in either luminal or bath perfusates. Thus both apical and basolateral CaSR on crypt cells are functional and provide pathways modulating net intestinal fluid transport that may have important implications for the prevention and treatment of certain diarrheal diseases associated with elevated cAMP.

ATP-sensitive potassium (K(ATP)) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K(ATP) channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5'-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH(2)- and COOH-terminal cytosolic regions of the three known K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K(+) channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K(ATP) channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K(ATP) channels because this nucleotide did not bind to the NH(2) termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K(ATP) COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K(ATP) COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg(2+) and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K(ATP) tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

Inwardly rectifying, ATP-sensitive K + channels (K ATP ) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P 2 , antagonize nucleotide inhibition of K ATP channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of K ATP channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of K ATP channels with EC 50 values for PIP 2 between 6–8 μM. The phospholipid potency profile was PIP 3 > PIP 2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP 2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP 2 , inositol(1,4,5)P 3 (IP 3 ), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP 2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP 2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP 2 for binding to these K ATP channel COOH termini. Thus the COOH termini of K ATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.

ATP-regulated (K(ATP)) channels are formed by an inward rectifier pore-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family (sulfonylurea receptor (SUR) or cystic fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to K(ATP) channels. In the mammalian kidney ROMK1-3 are components of K(ATP) channels that mediate K(+) secretion into urine. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH(2)-terminal extensions of 19 and 26 amino acids, respectively. The SUR2B is also expressed in rat kidney tubules and may combine with Kir.1 to form renal K(ATP) channels. Our previous studies showed that co-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive K(+) currents. These data suggest that the NH(2)-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH(2)-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH(2)-terminal deletion and substitution mutants and examined glibenclamide-sensitive K(+) currents in oocytes when co-expressed with SUR2B. These studies identified an amino acid triplet "IRA" within the conserved segment in the NH(2) terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to confer glibenclamide sensitivity to the expressed K(+) currents. The position of this triplet in the ROMK1 NH(2)-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation studies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct interaction between these two proteins is required for glibenclamide sensitivity of induced K(+) currents in oocytes. These results suggest that the IRA triplet in the NH(2)-terminal extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly of the renal secretary K(ATP) channel.

In the absence of vasopressin, medullary thick ascending limb cells express a K+-independent, furosemide-sensitive Na+-Cl−cotransporter that is inhibited by hypertonicity. The murine renal specific Na+-K+-2 Cl−cotransporter gene ( SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na+-K+-2 Cl−cotransporter (BSC1–9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (∼200 mosmol/kgH2O), mBSC1–4-injected oocytes do not express significant Na+uptake over H2O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1–4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1–4 oocytes are exposed to hypotonicity (∼100 mosmol/kgH2O), a significant increase in Na+uptake but not in86Rb+uptake is observed. The increased Na+uptake is Cl−dependent, furosemide sensitive, and cAMP sensitive but K+independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH2O ( r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na+-Cl−cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na+-Cl−-to-Na+-K+-2Cl−switching in thick ascending limb cells.

Dietary phosphate (Pi) intake and parathyroid hormone (PTH) are essential regulators of proximal tubular (PT) Pi reabsorption; both factors are associated with adaptive changes in PT apical brush border membrane (BBM) Na/Pi-cotransport activity and specific transporter protein (NaPi-2) content. Urinary Pi excretion is also inversely correlated with luminal Ca2+ concentration ([Ca2+]) both in a PTH-dependent and -independent fashion. A cell-surface, Ca2+(/polyvalent cation)-sensing receptor (CaR) has been localized to the PT BBM with unknown function. To investigate whether PTH and/or dietary Pi intake could affect the distribution or the expression of the CaR, we evaluated their effects on rat kidney CaR and the NaPi-2 expression by Western blot analysis and immunofluorescence microscopy. A chronic high-Pi (1.2%) versus low-Pi (0.1%) diet and acute PTH (1-34) infusion significantly reduced the PT BBM expression of both NaPi-2 and CaR proteins. CaR-specific immunoreactivity in nephron segments other than the PT was not affected by PTH or Pi intake. These results suggest that reduced renal PT CaR expression by a high-Pi diet and by increased circulating PTH levels could contribute to the local control of PT handling of Ca2+ and Pi.

To assess whether glucocorticoids regulate rBSC1, the apical Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter of kidney medullary thick ascending limb (MTAL), studies were performed in normal rats, adrenalectomized (ADX) rats, and ADX rats infused with dexamethasone for 6 days. The effects of dexamethasone on rBSC1 were also studied in vitro using isolated rat MTAL segments. Cotransport activity was estimated by intracellular pH measurements; rBSC1 protein was quantified in MTAL crude membranes by immunoblotting analysis, and mRNA was quantified by quantitative reverse transcription-polymerase chain reaction. The abundance of rBSC1 protein and mRNA increased in ADX rats infused with dexamethasone compared with ADX rats (p < 0. 04). In addition, application of dexamethasone for 1-3 h to MTALs caused rBSC1 protein and mRNA abundance and cotransport activity to significantly increase in a hyperosmotic medium (450 mosmol/kg of H(2)O) containing 0.7 nm arginine vasopressin, which is an in vitro experimental condition that resembles the in vivo MTAL environment. Results obtained in various media and with 8-bromo-cAMP indicated that stimulation of rBSC1 expression by glucocorticoids required interactions between glucocorticoid receptor- and cAMP-dependent factors. Up to 100 nm d-aldosterone had no effect on cotransport activity in vitro. Thus glucocorticoids directly stimulate MTAL rBSC1 expression and activity, which contributes to glucocorticoid-dependent effects on the renal regulation of acid-base balance and urinary concentrating ability.

Transforming growth factor β (TGF-β) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-β stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-β (2 μg/liter), phorbol 12,13-didecanoate (1 μM), or the Ca 2+ ionophore ionomycin (1 μM) and blunted by high concentrations of nifedipine (10 and 100 μM). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na + channel ENaC or the mouse thick ascending limb Na + ,K + ,2Cl − cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 μM) and chelerythrine (1 μM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na + transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.

The thiazide-sensitive Na+-Cl−cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant ( Km) for Na+of 7.6 ± 1.6 mM and for Cl−of 6.3 ± 1.1 mM, and Hill coefficients for Na+and Cl−consistent with electroneutrality. The affinities of both Na+and Cl−were increased by increasing concentration of the counterion. The IC50values for thiazides were affected by both extracellular Na+and Cl−. The higher the Na+or Cl−concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both Na+and Cl−.

The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl−cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of22Na+or86Rb+uptake. A total of six isoforms, generated by the combinatorial association of three 5′ exon cassettes (A, B, and F) with two alternative 3′ ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 ( Renal Physiol. 45): F347–F358, 1999]. The two 3′ ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl−cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl−cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl−cotransport in the mouse TAL by hormones that generate cAMP.

pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for p K = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. The K 1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to ∼0.5 mM at pH 7.2. Mutation of lysine-61 to methionine in ROMK2, which abolishes pH sensitivity, modulated but did not eliminate the effect of pH on ATP inhibition of channel activity. We previously demonstrated that the putative phosphate loop in the carboxy terminus of ROMK2 is involved in ATP binding and channel inhibition [C. M. McNicholas, Y. Yang, G. Giebisch, and S. C. Hebert. Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F275–F285, 1996]. Conceivably, therefore, protonation of the histidine residue within this region could alter net charge (i.e., positive shift) and increase affinity for the negatively charged nucleotide.

To assess whether metabolic acidosis per se regulates rBSC-1, the rat medullary thick ascending limb (MTAL) apical Na+-K+(NH4+)-2Cl- cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and membrane vesicles by intracellular pH and 22Na+ uptake measurements, respectively; rBSC-1 protein was quantified by immunoblotting analysis and mRNA by quantitative reverse transcription-polymerase chain reaction. As compared with incubation at pH approximately 7.35, acid incubation (pH approximately 7.10) up-regulated by 35-100% rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1 protein and mRNA abundance. In contrast, acid incubation did not alter alkaline phosphatase and Na+/K+-ATPase enzyme activities or beta-actin protein abundance. After 3 h of in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA abundance increased in freshly harvested MTALs, which was accompanied after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in vitro CMA stimulate rBSC-1 expression, which would contribute to the adaptive increase in MTAL absorption and urinary excretion of NH4+ in response to CMA.

The function of the apical Na+-K+-2Cl−cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl−cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl−/[Formula: see text](anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 μM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+removal or by addition of 100 μM bumetanide. Exposure of CP cells to 5 mM BaCl2induced rapid swelling that was inhibited by 100 μM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+channels to reabsorb K+from cerebrospinal fluid to blood.

The recent cloning, functional, morphologic, and genetic studies have established the CaSR as a vital component of the calcium homeostatic system. The CaSR provides both the sensing mechanism responsible for the regulation of parathyroid hormone secretion from parathyroid cells and the steep relationship between Ca2+ 0 and urinary calcium excretion in the kidney. The renal CaSR appears to provide the crucial “sensing” mechanism in the thick ascending limb and papillary collecting duct for integrating and balancing salt, water, and divalent mineral loss. Direct interactions of extracellular Ca2+ with the renal CaSR could explain in large part the disordered water metabolism (ie, nephrogenic diabetes insipidus) observed under pathologic states of hypercalcemia (eg, with primary hyperparathyroidism or associated with certain malignancies). The promise of addition of new calcimimetic agents to our therapeutic arsenal is an exciting prospect. The CaSR story provides an nice example of going from bench to bedside.

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K + secretory channels in the kidney. Addition of 1 μM forskolin or 100 μM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 μM forskolin, within 3 min, significantly increased whole-cell K + current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: ( i ) Addition of 100 μM 8-bromo-cAMP mimics the effect of forskolin and ( ii ) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 μM) and cAMP (100 μM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.

Electroneutral cation–chloride cotransporters are widely expressed and perform a variety of physiological roles. A novel gene family of five members, encompassing a Na+–Cl− transporter, two Na+–K+–2Cl− transporters and two K+–Cl− cotransporters, encodes these membrane proteins; homologous genes have also been identified in a prokaryote and a number of lower eukaryotes. The cotransporter proteins share a common predicted membrane topology, with twelve putative transmembrane segments flanked by long hydrophilic N- and C-terminal cytoplasmic domains. The molecular identification of these transporters has had a significant impact on the study of their function, regulation and pathophysiology.

Abstract —The purpose of the present study was to analyze the long-term regulation of renal bumetanide-sensitive Na + -K + -2Cl − cotransporter and thiazide-sensitive Na + -Cl − cotransporter gene expression during changes in NaCl and water metabolism. Male Wistar rats exposed to high or low NaCl intake, saline loading, dehydration, water loading, and furosemide administration during 7 days were studied. Control groups had access to regular food and tap water. Rats were kept in metabolic cages for 4 days before and during the experiment to determine daily urinary electrolyte excretion and osmolarity. At the end of the experiment, creatinine clearance and serum electrolyte levels were also measured. Kidneys were excised and macroscopically subdivided into cortex and outer and inner medulla. Total RNA was extracted from each individual cortex or outer medulla by use of the guanidine/cesium chloride method. The Na + -K + -2Cl − cotransporter expression in outer medulla total RNA was assessed by nonradioactive Northern blot analysis and the Na + -Cl − cotransporter expression in renal cortex total RNA was assessed by semiquantitative polymerase chain reaction. Experimental maneuvers were adequately tolerated, and all groups developed the appropriate renal response to each challenge. However, the level of expression of both cotransporters did not change in any model, except for a 2.8-fold increase in the Na + -Cl − cotransporter expression during dehydration. We conclude that nephron adaptation to 7-day modifications in NaCl and water metabolism does not include changes in the amount of electroneutral sodium-coupled cotransporter gene expression at the mRNA level.

The recent cloning of an extracellular calcium (Ca2+o)-sensing receptor (CaR) from parathyroid, kidney and other cell types has clarified the mechanisms through which Ca2+o exerts its direct actions on various cells and tissues. In the parathyroid, the CaR mediates the inhibitory effects of Ca2+o on parathyroid hormone (PTH) secretion and likely on expression of the PTH gene and parathyroid cellular proliferation. In the kidney, the receptor mediates direct inhibition of the reabsorption of divalent cations in the cortical thick ascending limb, and it likely underlies the inhibitory actions of hypercalcemia on the urinary-concentrating mechanism in the medullary thick ascending limb and inner medullary collecting duct. The identification of inherited diseases of Ca2+o-sensing that arise from mutations in the CaR gene has proven, by genetic means, the central role of the CaR in mineral ion homeostasis and the importance of the receptor in regulating the parathyroid and kidney. An allosteric CaR agonist (“calcimimetic”) is currently being tested for the treatment of primary hyperparathyroidism, and CaR-based therapeutics will likely be applicable to other disorders in which CaRs are under- or overactive. Thus the discovery of the CaR and its associated diseases has documented that Ca2+o plays an essential role as an extracellular first messenger, in addition to serving its better recognized role as an intracellular second messenger.

The extracellular calcium ([Formula: see text])-sensing receptor (CaR) plays vital roles in [Formula: see text] homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from reverse-transcribed duodenal RNA using CaR-specific primers showed >99% homology with the rat kidney CaR. Northern analysis with a CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all intestinal segments. Immunohistochemistry with CaR-specific antisera showed clear basal staining of epithelial cells of small intestinal villi and crypts and modest apical staining of the former, whereas there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach’s myenteric plexus of small and large intestines and in the submucosa in the region of Meissner’s plexus. Our results reveal CaR expression in several cell types of small and large intestine, in which it may modulate absorptive and/or secretomotor functions.

An inhibitory neurotransmitter in mature brain, gamma-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl- accumulation in immature neurons and oligodendrocytes.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca2+-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

The ATP-sensitive, inwardly rectifying K+channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H+-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+secretory channel in the rat distal nephron.

In a previous study on inside-out patches of Xenopus oocytes, we demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) enhances the glibenclamide sensitivity of a coexpressed inwardly rectifying K+ channel, ROMK2 (C. M. McNicholas, W. B. Guggino, E. M. Schwiebert, S. C. Hebert, G. Giebisch, and M. E. Egan. Proc. Natl. Acad. Sci. USA 93: 8083–8088, 1996). In the present study, we used the two-microelectrode voltage-clamp technique to measure whole cell K+ currents in Xenopus oocytes, and we further characterized the enhanced sensitivity of ROMK2 to glibenclamide by CFTR. Glibenclamide inhibited K+currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11% in oocytes expressing ROMK2 alone. To examine the role of the first nucleotide binding fold (NBF1) of CFTR in the ROMK2-CFTR interaction, we studied the glibenclamide sensitivity of ROMK2 when coexpressed with CFTR constructs containing mutations in or around the NBF1 domain. In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants with naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E, display glibenclamide-inhibitable K+currents of only 14 and 25%, respectively. Because CFTR mutations that alter the NBF1 domain reduce the glibenclamide sensitivity of the coexpressed ROMK2 channel, we conclude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity.

▪ Abstract The activity of potassium (K+) channels is intimately linked to several important transport functions in renal tubules. We review recent progress concerning the properties, site along the nephron, and physiological regulation of native K+ channels, and compare their characteristics with those of recently cloned K+ channels. We do not fully cover work on K+ channels in amphibian tubules, cell cultures, and single tubule cells and do not review K+ channels in mesangial cells.

The application of molecular biology to the study of electroneutral cation-chloride cotransporters has been extremely successful, resulting in the identification of a new gene family of five membrane proteins. The function, expression, and regulation of these important proteins can increasingly be described in molecular terms. In addition, mutations in two renal cation-chloride transporter genes have been found in patients with Bartter's and Gitelman's syndromes, autosomal recessive disorders of renal salt excretion.

The divalent mineral cations Ca2+ and Mg2+ play many and diverse roles both in the function of cells and in extracellular processes. The metabolism of these cations is a complex process involving the coordinated function of several organ systems and endocrine glands. A recently cloned G-protein-coupled receptor responds to extracellular calcium concentration (Ca2+o-sensing receptor, CaSR) and mediates several of the known effects of Ca2+o on parathyroid and renal function. The CaSR, which is also expressed in a number of other tissues including thyroidal C-cells, brain and gastrointestinal tract, may function as a Ca2+o sensor in these tissues as well. Thus, Ca2+o is a first messenger (or hormone) which, via CaSR-mediated activation of second messenger systems (e.g. phospholipases C and A2, cyclic AMP) leads to altered function of these cells. Several mutations in the human CaSR gene have been identified and shown to cause three inherited diseases of calcium homeostasis, clearly implicating the CaSR as an important component of the homeostatic mechanism for divalent mineral ions. Ca2+ and Mg2+ losses from the body are regulated by altering the urinary excretion of these divalent cations. The localization of the CaSR transcripts and protein in the kidney not only provides a basis for a direct Ca2+o (or Mg2+o) -mediated regulation of Ca2+ (and Mg2+) excretion but also suggests a functional link between divalent mineral and water metabolism. In the kidney, the thick ascending limb of Henle (TAL) plays crucial roles in regulating both divalent mineral reabsorption and urine concentration. Recent studies have suggested models whereby extracellular Ca2+, via the CaSR expressed in the TAL as well as in the collecting duct system, modulates both Ca2+o and Mg2+o as well as water reabsorbtion. When taken together, these studies suggest that the CaSR not only provides the primary mechanism for Ca2+o-mediated regulation of parathyroid hormone secretion from parathyroid glands but also for direct modulation of renal divalent mineral excretion and urinary concentrating ability. These latter functions may furnish a mechanism for integrating and balancing water and divalent cation losses that minimizes the risk of urinary tract stone formation. This mechanism can explain hypercalcemia-mediated polyuria (diabetes insipidus).

The capacity to sense changes in the concentrations of extracellular ions is an important function in several cell types. For example, hormone secretion by parathyroid cells and thyroid C-cells is primarily regulated by the level of extracellular ionized calcium (Ca2+). The G-protein-coupled receptor that mediates the parathyroid cell response to Ca2+ has been cloned and we have used in situ hybridization to map calcium receptor (CaR) mRNA expression in the adult rat brain. Cells expressing CaR mRNA were present in many areas of the brain suggesting that a variety of cell types express the CaR. Particularly high numbers of CaR expressing cells were found in regions associated with the regulation of fluid and mineral homeostasis, most notably the subfornical organ. These data suggest that the capacity to detect changes in extracellular Ca2+ concentrations may have important functional consequences in several neural systems.

The acute secretory response of parathyroid hormone (PTH) is strictly regulated by the extracellular calcium concentration (Ca2+ o), and the G protein-coupled, calcium-sensing receptor (CaR) located on the chief cells of the parathyroid glands mediates this process. Abnormalities of the Ca2+ o-sensing system lead to diseases that show hypo-/hypersecretion of PTH in addition to relative hyper-/hypocalciuria. Novel signaling pathways, e.g., mitogen-activated protein kinases (MAPK), have been shown to be involved in CaR signaling in addition to “classical” CaR-regulated pathways, e.g., phospholipase C (PLC) and adenylate cyclase. We will discuss the following topics in this chapter: (1) the regulatory mechanisms of Ca2+ o-sensing and PTH secretion, (2) disorders due to mutations in the CaR gene, abnormal CaR expression, or the production of antibodies against the CaR, and (3) the promising utility of drugs acting on the CaR.