scholarly journals ENaC activity is regulated by calpain-2 proteolysis of MARCKS proteins

2017 ◽  
Vol 313 (1) ◽  
pp. C42-C53 ◽  
Author(s):  
Darrice S. Montgomery ◽  
Ling Yu ◽  
Zinah M. Ghazi ◽  
Tiffany L. Thai ◽  
Otor Al-Khalili ◽  
...  
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
Adaptor Protein ◽  
Cysteine Proteases ◽  
Cortical Collecting Duct ◽  
Anionic Phospholipid ◽  
Kinase Substrate ◽  
Recombinant Fusion Proteins ◽  
Intracellular Ca ◽  
Calpain 2

We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca2+. Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.

scholarly journals Mal protein stabilizes luminal membrane PLC-β3 and negatively regulates ENaC in mouse cortical collecting duct cells

2019 ◽  
Vol 317 (4) ◽  
pp. F986-F995
Author(s):  
Kubra M. Tuna ◽  
Bing-Chen Liu ◽  
Qiang Yue ◽  
Zinah M. Ghazi ◽  
He-Ping Ma ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Expression ◽  
Collecting Duct ◽  
Renal Tubules ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
Anionic Phospholipid ◽  
Kinase Substrate ◽  
Luminal Membrane ◽  
Collecting Duct Cells

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl− cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

Adenosine-stimulated Ca2+reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system

1998 ◽  
Vol 274 (4) ◽  
pp. F736-F743 ◽  
Author(s):  
Joost G. J. Hoenderop ◽  
Anita Hartog ◽  
Peter H. G. M. Willems ◽  
René J. M. Bindels
Keyword(s):  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Concomitant Increase ◽  
Cgs 21680 ◽  
Kinase C ◽  
Intracellular Ca ◽  
Collecting Duct Cells ◽  
Camp Formation ◽  
Pkc Activation ◽  
Permeable Supports

Confluent monolayers of immunodissected rabbit connecting tubule and cortical collecting duct cells, cultured on permeable supports, were used to study the effect of adenosine on net apical-to-basolateral Ca2+ transport. Apical, but not basolateral, adenosine increased this transport dose dependently from 48 ± 3 to 110 ± 4 nmol ⋅ h−1 ⋅ cm−2. Although a concomitant increase in cAMP formation suggested the involvement of an A2 receptor, the A2 agonist CGS-21680 did not stimulate Ca2+ transport, while readily increasing cAMP. By contrast, the A1 agonist N 6-cyclopentyladenosine (CPA) maximally stimulated Ca2+transport without significantly affecting cAMP. Adenosine-stimulated transport was effectively inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopenthylxanthine but not the A2 antagonist 3,7-dimethyl-1-propargylxanthine, providing additional evidence for the involvement of an A1 receptor. Both abolishment of the adenosine-induced transient increase in intracellular Ca2+ concentration by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid and downregulation of protein kinase C (PKC) by prolonged phorbol ester treatment were without effect on adenosine-stimulated Ca2+ transport. The data presented suggest that adenosine interacts with an apical A1 receptor to stimulate Ca2+ transport via a hitherto unknown pathway that does not involve cAMP formation, PKC activation, and/or Ca2+ mobilization.

Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture

2001 ◽  
Vol 280 (5) ◽  
pp. C1193-C1203 ◽  
Author(s):  
Laura Valencia ◽  
Michel Bidet ◽  
Sonia Martial ◽  
Elsa Sanchez ◽  
Estela Melendez ◽  
...  
Keyword(s):  
Primary Culture ◽  
Transient Receptor Potential ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Receptor Potential ◽  
Cortical Collecting Duct ◽  
Newborn Rat ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Transient Receptor

To characterize Ca2+ transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca2+concentration ([Ca2+]i) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 μM) produced an increase in [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca2+]i in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca2+]i. Experiments in the presence of EGTA showed that external Ca2+ was required for the nifedipine effect, while lanthanum (20 μM), gadolinium (100 μM), and diltiazem (20 μM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K+ channels were not involved in the nifedipine-induced [Ca2+]i rise. H2O2also triggered [Ca2+]i rise. However, nifedipine-induced [Ca2+]i increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca2+transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca2+ channel of capacitive type (either transient receptor potential or leak channel).

scholarly journals ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl− secretion

2015 ◽  
Vol 309 (3) ◽  
pp. F251-F258 ◽  
Author(s):  
Masayoshi Nanami ◽  
Yoskaly Lazo-Fernandez ◽  
Vladimir Pech ◽  
Jill W. Verlander ◽  
Diana Agazatian ◽  
...  
Keyword(s):  
Channel Blocker ◽  
Single Channel ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Collecting Ducts ◽  
Hcl Secretion ◽  
Transepithelial Voltage ◽  
Apical Membranes ◽  
Rats And Mice

Inhibition of the epithelial Na+ channel (ENaC) reduces Cl− absorption in cortical collecting ducts (CCDs) from aldosterone-treated rats and mice. Since ENaC does not transport Cl−, the purpose of the present study was to explore how ENaC modulates Cl− absorption in mouse CCDs perfused in vitro. Therefore, we measured transepithelial Cl− flux and transepithelial voltage in CCDs perfused in vitro taken from mice that consumed a NaCl-replete diet alone or the diet with aldosterone administered by minipump. We observed that application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid unmasks conductive Cl− secretion. During ENaC blockade, this Cl− secretion fell with the application of a nonselective Cl− channel blocker [DIDS (100 μM)] to the perfusate. While single channel recordings of intercalated cell apical membranes in split-open CCDs demonstrated a Cl− channel with properties that resemble the ClC family of Cl− channels, ClC-5 is not the primary pathway for benzamil-sensitive Cl− flux. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl− absorption is benzamil sensitive, and, second, benzamil application stimulates stilbene-sensitive conductive Cl− secretion, which occurs through a ClC-5-independent pathway.

scholarly journals Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD)

2012 ◽  
Vol 302 (1) ◽  
pp. F205-F215 ◽  
Author(s):  
Wen Liu ◽  
Núria M. Pastor-Soler ◽  
Carlos Schreck ◽  
Beth Zavilowitz ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Axial Flow ◽  
Bk Channel ◽  
Cortical Collecting Duct ◽  
Dolichos Biflorus ◽  
Intracellular Ca ◽  
Acid Load ◽  
Luminal Flow ◽  
Principal And Intercalated Cells

Epithelial Na+ channel (ENaC)-mediated Na+ absorption and BK channel-mediated K+ secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca2+ concentration ([Ca2+]i), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO3− reabsorption in the proximal tubule due to changes in both luminal Na+/H+ exchanger 3 and H+-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289–F296, 2006), we sought to test the hypothesis that flow also regulates H+-ATPase activity in the CCD. H+-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH4Cl prepulse technique). H+-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pHi) recovery in the absence of luminal K+, bilateral Na+, and CO2/HCO3−, from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min−1·mm−1 stimulated H+-ATPase activity, 2) flow-stimulated H+ pumping was Ca2+ dependent and required microtubule integrity, and 3) basal and flow-stimulated pHi recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H+-ATPase activity in the rabbit CCD and that H+-ATPases therein are present in both principal and intercalated cells.

scholarly journals Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

2012 ◽  
Vol 302 (6) ◽  
pp. F679-F687 ◽  
Author(s):  
Peng Sun ◽  
Peng Yue ◽  
Wen-Hui Wang
Keyword(s):  
Angiotensin Ii ◽  
Single Channel ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Whole Cell ◽  
Na Currents

We examined the effect of angiotensin II (ANG II) on epithelial Na+channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NPo(a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca2+with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca2+-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H2O2diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca2+-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.

scholarly journals Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage

2016 ◽  
Vol 310 (4) ◽  
pp. F311-F321 ◽  
Author(s):  
Oleg Zaika ◽  
Oleg Palygin ◽  
Viktor Tomilin ◽  
Mykola Mamenko ◽  
Alexander Staruschenko ◽  
...  
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Sodium Reabsorption ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
Whole Cell ◽  
Principal Cells ◽  
Basolateral Side ◽  
Kinase Cascade

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K+-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na+-K+-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K+-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na+ reabsorption in the CCD.

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron

2012 ◽  
Vol 303 (9) ◽  
pp. F1289-F1299 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Anke Dahlmann ◽  
Bettina Krueger ◽  
Marko Bertog ◽  
Johannes Loffing ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Salt Diet ◽  
Low Salt ◽  
Immunohistochemical Evidence ◽  
Epithelial Sodium Channel Enac

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na+ channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (Δ Iami) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased Δ Iami in CNT/CCD but had little effect on Δ Iami in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS−/−) mice that lack aldosterone. In CNT/CCD of AS−/− mice, Δ Iami was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS−/− mice, Δ Iami was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

Cloning and functional expression of the mouse epithelial sodium channel

1999 ◽  
Vol 277 (1) ◽  
pp. F121-F129 ◽  
Author(s):  
Yoon J. Ahn ◽  
David R. Brooker ◽  
Farhad Kosari ◽  
Brian J. Harte ◽  
Jinqing Li ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Distal Colon ◽  
Dominant Negative ◽  
Full Length ◽  
Cortical Collecting Duct ◽  
Race Pcr

The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed α-, β-, and γENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse α-, β-, and γENaC (α-, β-, and γmENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length βmENaC and partial-length α- and γmENaC clones were isolated. Full-length α- and γmENaC cDNAs were subsequently obtained by 5′-rapid amplification of cDNA ends (5′-RACE) PCR. Injection of mouse α-, β-, and γENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive ( K i = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that α-, β-, and γmENaC were expressed in lung and kidney. Interestingly, αmENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5′-RACE PCR. Its sequence was found to be nearly identical to αmENaC. To begin to identify regions within αmENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of αmENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type α-, β-, and γmENaC with the intracellular NH2 terminus of αmENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of αmENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.

Contrasting effects of cPLA2 on epithelial Na+ transport

2001 ◽  
Vol 281 (1) ◽  
pp. C147-C156 ◽  
Author(s):  
Roger T. Worrell ◽  
Hui-Fang Bao ◽  
Don D. Denson ◽  
Douglas C. Eaton
Keyword(s):  
Aristolochic Acid ◽  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Fine Tuning ◽  
Prostaglandin E ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
A6 Cells ◽  
Metabolic Products

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+reabsorption in the cortical collecting duct. Xenopus laeviskidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2(PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.

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