Apical Entry Research Articles

Role of the Transient Receptor Potential Vanilloid 5 (TRPV5) Protein N Terminus in Channel Activity, Tetramerization, and Trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.

Mechanism of action of 1,25-dihydroxyvitamin D3 on intestinal calcium absorption

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is the major controlling hormone of intestinal calcium absorption. As the body's demand for calcium increases from a diet deficient in calcium, from growth, pregnancy or lactation, the synthesis of 1,25(OH)(2)D(3) is increased resulting in the stimulation of intestinal calcium absorption. However a complete description of the molecular mechanisms involved in the 1,25(OH)(2)D(3) regulated calcium absorptive process remains incomplete. Intestinal calcium absorption occurs by both an active saturable transcellular pathway and a passive nonsaturable paracellular pathway. Each step in the process of transcellular calcium transport (apical entry of calcium, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the plasma membrane pump) has been reported to involve a vitamin D dependent component. This article will review recent studies, including those using knockout mice, that have suggested that 1,25(OH)(2)D(3) mediated calcium absorption is more complex than the traditional three step model of transcellular calcium transport. Current concepts are reviewed and questions that remain are addressed. Evidence for a role of 1,25(OH)(2)D(3) in the regulation of the paracellular pathway is also discussed.

Chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells

Mucosal epithelia provide strong barriers against pathogens. For instance, the outward facing apical membrane of polarized epithelial cells lacks receptors for agents, such as hepatitis C virus, herpesvirus, reovirus, poliovirus or adenovirus. In addition, macrophages eliminate pathogens from the luminal space. Here we show that human adenovirus type 5 engages an antiviral immune response to enter polarized epithelial cells. Blood-derived macrophages co-cultured apically on polarized epithelial cells facilitate epithelial infection. Infection also occurs in the absence of macrophages, if virus-conditioned macrophage-medium containing the chemotactic cytokine CXCL8 (interleukin-8), or recombinant CXCL8 are present. In polarized cells, CXCL8 activates a Src-family tyrosine kinase via the apical CXCR1 and CXCR2 receptors. This activation process relocates the viral co-receptor ανβ3 integrin to the apical surface, and enables apical binding and infection with adenovirus depending on the primary adenovirus receptor CAR. This paradigm may explain how other mucosal pathogens enter epithelial cells.Supplementary informationThe online version of this article (doi:10.1038/ncomms1391) contains supplementary material, which is available to authorized users.

Molecular Mechanisms of Calmodulin Action on TRPV5 and Modulation by Parathyroid Hormone

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca(2+) reabsorption in the kidney. Ca(2+) influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca(2+) influx. This inactivation is mediated by the last ∼30 residues of the carboxy (C) terminus of the channel. Since the Ca(2+)-sensing protein calmodulin has been implicated in Ca(2+)-dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca(2+)-dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca(2+)-dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

Aminoglycosides Inhibit KCNQ4 Channels in Cochlear Outer Hair Cells via Depletion of Phosphatidylinositol(4,5)bisphosphate

Aminoglycoside antibiotics (AGs) are severely ototoxic. AGs cause degeneration of outer hair cells (OHCs), leading to profound and irreversible hearing loss. The underlying mechanisms are not fully understood. OHC survival critically depends on a specific K+ conductance (I(K,n)) mediated by KCNQ4 (Kv7.4) channels. Dysfunction or genetic ablation of KCNQ4 results in OHC degeneration and deafness in mouse and humans. As a common hallmark of all KCNQ isoforms, channel activity requires phosphatidylinositol(4,5)bisphosphate [PI(4,5)P₂]. Because AGs are known to reduce PI(4,5)P₂ availability by sequestration, inhibition of KCNQ4 may be involved in the action of AGs on OHCs. Using whole-cell patch-clamp recordings from rat OHCs, we found that intracellularly applied AGs inhibit I(K,n). The inhibition results from PI(4,5)P₂ depletion indicated by fluorescence imaging of cellular PI(4,5)P₂ and the dependence of inhibition on PI(4,5)P₂ availability and on PI(4,5)P₂ affinity of recombinant KCNQ channels. Likewise, extracellularly applied AGs inhibited I(K,n) and caused substantial depolarization of OHCs, after rapid accumulation in OHCs via a hair cell-specific apical entry pathway. The potency for PI(4,5)P₂ sequestration, strength of I(K,n) inhibition, and resulting depolarization correlated with the known ototoxic potential of the different AGs. Thus, the inhibition of I(K,n) via PI(4,5)P₂ depletion and the resulting depolarization may contribute to AG-induced OHC degeneration. The KCNQ channel openers retigabine and zinc pyrithione rescued KCNQ4/I(K,n) activity from AG-induced inhibition. Pharmacological enhancement of KCNQ4 may thus offer a protective strategy against AG-induced ototoxicity and possibly other ototoxic insults.

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura-2 analysis. Removal of amino acid His(712) elevated the [Ca(2+)](i), indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His(712) for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His(712) was confirmed by patch clamp analysis, which demonstrates increased Na(+) and Ca(2+) currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca(2+)-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca(2+)](i). Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His(712) plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.

Adenoviral Transduction of Enterocytes and M-Cells Using in Vitro Models Based on Caco-2 Cells: The Coxsackievirus and Adenovirus Receptor (CAR) Mediates Both Apical and Basolateral Transduction

Understanding virus-cell interaction is a key to the design of successful gene delivery vectors. In the present study we investigated Ad5 transduction of enterocytes and M-cells utilizing differentiated Caco-2 cells and cocultures of Caco-2 cells with lymphocytes. Transduction inhibition studies showed that CAR is the major receptor mediating apical and basolateral virus entry in differentiated Caco-2 cells. Integrins and heparan sulfate glycosaminoglycans do not appear to play a significant role. Immunofluorescence localized CAR to sites of cell-cell contact, with staining mostly observed on the cell perimeter. Staining was observed even in nonpermeabilized monolayers, suggesting apical accessibility of the receptor. Cocultures with mouse Peyer's patch lymphocytes or Raji B human lymphocytes were more susceptible to transduction than Caco-2 cells, and the effects were dose-dependent. Similar to Caco-2 cells, CAR and not integrins mediated apical transduction. In conclusion, contrary to other epithelial cell lines, both apical and basolateral transduction of absorptive enterocytes and M-cells is mediated by binding to CAR. The coculture system can be used to study the interactions between M-cells and gene delivery vectors.

Insulin down-regulates the Na +/K + ATPase in enterocytes but increases intestinal glucose absorption

The effect of insulin on [ 14C] 3-O-methyl- d-glucose (3OMG) absorption in the rat jejunum was studied using an in situ perfusion technique. Insulin increased apical glucose entry into the cells and decreased intestinal retention suggesting that serosal glucose transport was enhanced by the hormone. This enhanced uptake was ascribed to an increase in the expression of glucose transporters as confirmed by Western blot analysis and not to a higher sodium gradient, since insulin reduced the activity and protein expression of the Na +/K + ATPase. To separate the glycemic from the insulinemic effect on glucose transport, the effect of the hormone was investigated in vitro using cultured Caco-2 cells. The cells also showed an increase in [ 14C] 3OMG uptake and intracellular glucose levels when treated with insulin and a lower Na +/K + ATPase activity. Phloretin, an inhibitor of GLUT2 was used to determine if these transporters are targeted by the hormone. The results showed that the effect of insulin on glucose uptake and intracellular glucose was still enhanced in presence of phloretin. Considering the inhibitory effect of the hormone on the Na +/K + ATPase, it was concluded that insulin acts by increasing the number of glucose transporters, a hypothesis that was confirmed by Western blot analysis.

Thiazide Effects and Adverse Effects

One of the longest-running debates in clinical medicine shows no sign of disappearing; just when it seems that thiazides have reassumed their role as front-line drugs to treat hypertension,1 new concerns emerge,2–4 leading some to question their role once again.5 Thiazides are effective antihypertensives with long track records and low cost. The major concerns about their use arise from their tendency to cause hypokalemia, impair glucose tolerance, increase serum cholesterol, and increase serum uric acid. Few medical controversies have generated as much heat, with well-established camps staking out positions that appear resistant to change.6–9 The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial, the largest study of antihypertensive monotherapy ever performed,10 was intended to identify the best first-line treatment for high-risk hypertensive individuals; however, despite its size and the numerous resulting publications, its implications and authority continue to be disputed. The goal of this review is not take sides in this debate but rather to inject a distinct, and sometimes neglected, perspective; during the past 15 years, remarkable developments in molecular biology and human genetics have provided substantial insights into the pathogenesis of hypertension and mechanisms and adverse effects of diuretics. Diuretic proponents and antagonists alike often neglect these developments when addressing the topic; it is the purpose of this Brief Review to integrate these developments into the debate with the goal of generating questions that can be addressed scientifically. Thiazide diuretics were developed during the 1950s, when chemists and physiologists at Merck Sharpe and Dohme tested derivatives of sulfonamide-based carbonic anhydrase inhibitors, with the goal of discovering drugs that enhance the excretion of sodium with chloride rather than sodium bicarbonate.* Although these drugs lower arterial pressure effectively, the mechanisms have long perplexed investigators.11 Thiazides reduce cardiac output acutely by reducing extracellular …

Enhancement of calcium transport in Caco-2 monolayer through PKCζ-dependent Cav1.3-mediated transcellular and rectifying paracellular pathways by prolactin

Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca(2+)-ATPase (PMCA) inhibitor (trifluoperazine) as well as small interfering RNA targeting voltage-dependent L-type calcium channel Ca(v)1.3, but not TRPV6 or calbindin-D(9k). As demonstrated by (45)Ca uptake study, PI3K and PKC, but not ROCK, were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKC(zeta) knockdown or exposure to inhibitors of PKC(zeta), but not of PKC(alpha), PKC(beta), PKC(epsilon), PKC(mu), or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY-294002) and ROCK (Y-27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Ca(v)1.3 and PMCA, presumably through PI3K and PKC(zeta) pathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.

Activation of the Ca 2+-sensing receptor stimulates the activity of the epithelial Ca 2+ channel TRPV5

The extracellular Ca 2+-sensing receptor (CaR) is a key-player in plasma Ca 2+ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca 2+ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca 2+-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca 2+ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca 2+ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca 2+ influx via a PMA-insensitive PKC isoform pathway.

Active Ca2+ reabsorption in the connecting tubule

The kidney plays a crucial role in the maintenance of the body calcium (Ca(2+)) balance. Ca(2+) is an essential ion in all organisms and participates in a large variety of structural and functional processes. In mammals, active tubular Ca(2+) reabsorption is restricted to the distal part of the nephron, i.e., the late distal convoluted (DCT2) and the connecting tubules (CNT), where approximately 10-15% of the total Ca(2+) is reabsorbed. This active transcellular transport is hallmarked by the transient receptor potential vanilloid 5 (TRPV5) epithelial Ca(2+) channel, regulated by an array of events, and mediated by hormones, including 1,25-dihydroxyvitamin D(3), parathyroid hormone, and estrogen. Novel molecular mechanisms have been identified, such as the direct regulatory effects of klotho and tissue kallikrein on the abundance of TRPV5 at the apical membrane. The newly discovered mechanisms could provide potential pharmacological targets in the therapy of renal Ca(2+) wasting. This review discusses the three basic molecular steps of active Ca(2+) reabsorption in the DCT/CNT segments of the nephron, including apical entry, cytoplasmic transport, and basolateral extrusion of Ca(2+). In addition, an overview of the recently identified mechanisms governing this active Ca(2+) transport through the DCT2/CNT epithelial cells will be presented.

WNK4 regulates the secretory pathway via which TRPV5 is targeted to the plasma membrane

TRPV5 and TRPV6 are two closely related epithelial calcium channels that mediate apical calcium entry in the transcellular calcium transport pathway. TRPV5, but not TRPV6, is enhanced by protein kinase WNK4 when expressed in Xenopus laevis oocytes. We report that the majority of human TRPV5 exogenously expressed in the Xenopus oocyte plasma membrane was complexly N-glycosylated whereas that for human TRPV6 was core-glycosylated. Unglycosylated N358Q mutants of TRPV5 and TRPV6 were able to be expressed in the plasma membrane albeit with decreased abilities in mediating calcium uptake. Syntaxin 6, a SNARE protein in the trans-Golgi network, blocked the complex glycosylation of TRPV5 and TRPV6, rendered the channels in core-glycosylated form. Blocking complex glycosylation of TRPV5 either by syntaxin 6 or by N358Q mutation abolished the enhancing effect of WNK4 on TRPV5. Thus the difference in membrane expression of TRPV5 and TRPV6 explains the selective effect of WNK4 on TRPV5, which is likely on the secretory pathway involving complex glycosylation of channel proteins.

Molecular Determinants of Magnesium Homeostasis

The past decade has witnessed multiple advances in our understanding of magnesium (Mg(2+)) homeostasis. The discovery that mutations in claudin-16/paracellin-1 or claudin-19 are responsible for familial hypomagnesemia with hypercalciuria and nephrocalcinosis provided insight into the molecular mechanisms governing paracellular transport of Mg(2+). Our understanding of the transcellular movement of Mg(2+) was similarly enhanced by the realization that defects in transient receptor potential melastatin 6 (TRPM6) cause hypomagnesemia with secondary hypocalcemia. This channel regulates the apical entry of Mg(2+) into epithelia. In so doing, TRPM6 alters whole-body Mg(2+) homeostasis by controlling urinary excretion. Consequently, investigation into the regulation of TRPM6 has increased. Acid-base status, 17beta estradiol, and the immunosuppressive agents FK506 and cyclosporine affect plasma Mg(2+) levels by altering TRPM6 expression. A mutation in epithelial growth factor is responsible for isolated autosomal recessive hypomagnesemia, and epithelial growth factor activates TRPM6. A defect in the gamma-subunit of the Na,K-ATPase causes isolated dominant hypomagnesemia by altering TRPM6 activity through a decrease in the driving force for apical Mg(2+) influx. We anticipate that the next decade will provide further detail into the control of the gatekeeper TRPM6 and, therefore, overall whole-body Mg(2+) balance.

Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene (Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct

Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry.

NKCC1 Is Phosphorylated in Rat Inner Medullary Collecting Duct (IMCD)

Previously, we demonstrated a bumetanide‐inhibitable increase in IMCD cell height in response to vasopressin (AVP) in isolated perfused tubules in which the bath osmolality was increased to 490 mOsm (lumen 290 mOsm). This suggests that AVP induced cell swelling was not due to apical water entry, but rather basolateral solute uptake via NKCC1, followed by water entry. To address the role of NKCC1 we used an N‐terminal directed antibody recognizing total NKCC1 (α‐NT) and a phospho‐directed antibody targeted to Thr‐212 and Thr‐217 in the N‐terminal tail of human NKCC1 (R5). Using the α‐NT antibody for immunofluorescence, we confirmed that NKCC1 is localized to the basolateral membrane of the rat IMCD cell. To test if NKCC1 is phosphorylated, we used the R5 phospho‐antibody in immunoblotting. The antibody recognized 170 and 150 kDa bands in IMCD suspensions under control conditions (290 mOsm). Hypertonicity (490 mOsm) caused the band density of the lower band to increase by 2‐fold (P<0.05, n=3). There was no obvious change in mobility of proteins recognized by a COOH‐tail antibody. A dephosphorylation experiment using λ‐phosphatase confirmed that the R5 antibody recognized only phosphorylated proteins. AVP alone did not change the band pattern with the R5 antibody. We conclude that in IMCD cells NKCC1 is a phospho‐protein and that hypertonicity affects the mobility or phosphorylation state of the NKCC1 phosphoprotein.

Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca(2+) reabsorption in kidney. Many hormones alter renal Ca(2+) reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca(2+) reabsorption by PTH.

Renal magnification by EGF

A paper recently published by Groenestege and colleagues [8] used positional cloning to determine the cause of a rare inherited magnesium-wasting syndrome, autosomal recessive renal hypomagnesemia. The results showed that certain mutations in the epidermal growth factor (EGF) gene cause this disease. This suggests, perhaps surprisingly, that EGF and its receptor comprise a previously unrecognized signaling pathway in the human kidney that participates importantly in magnesium homeostasis. The EGF gene is highly expressed along the distal convoluted tubule (DCT), an important site for regulating urinary magnesium excretion. The product of the gene is a large membrane-bound molecule, expressed at both the apical and basolateral surfaces. A portion of the extracellular domain can be cleaved to form the 53-amino-acid hormone, EGF; it is believed that cleavage occurs preferentially at the basolateral surface, leading to increased apparent EGF abundance at the apical membrane. In the DCT, EGF binds to receptors at the basolateral surface, making basolateral EGF expression a necessary prerequisite for signaling. The gene defect that causes autosomal recessive renal hypomagnesemia appears to disrupt trafficking of pro-EGF to the basolateral membrane, thereby impeding its signaling capability. The results also showed that EGF stimulates magnesium transport by TRPM6 (transient receptor potential cation channel, subfamily M, member 6), a channel that is expressed at the apical membrane of DCT cells and appears to be a primary path for apical magnesium entry. Interestingly, the investigators also corroborated previous reports that cancer patients treated with an antagonist of the EGF receptor, cetuximab, develop renal magnesium wasting and hypomagnesemia, emphasizing the significance of EGF in maintaining magnesium balance in humans.