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.

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.

Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct

1995 ◽  
Vol 269 (3) ◽  
pp. C791-C796 ◽  
Author(s):  
J. K. Bubien
Keyword(s):  
Patch Clamp ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Principal Cells ◽  
Clamp Technique ◽  
Duct Cells ◽  
Collecting Duct Cells

Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats. The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640. Individual cells were examined electrophysiologically using the whole cell patch-clamp technique. Two morphologically distinct cell types were present in the cell suspension. Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed. Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells. Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells. The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor). These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells. Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.

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.

scholarly journals Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells

2016 ◽  
Vol 311 (6) ◽  
pp. F1360-F1368 ◽  
Author(s):  
Ming-Ming Wu ◽  
Yu-Jia Zhai ◽  
Yu-Xia Li ◽  
Qing-Qing Hu ◽  
Zhi-Rui Wang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Apical Membrane ◽  
Intact Cell ◽  
Collecting Duct ◽  
Physiological Role ◽  
Cell Types ◽  
Cortical Collecting Duct ◽  
Principal Cells

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

scholarly journals Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells.

1996 ◽  
Vol 108 (3) ◽  
pp. 177-193 ◽  
Author(s):  
K Meyer ◽  
C Korbmacher
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Collecting Duct ◽  
High Energy ◽  
Cell Swelling ◽  
Single Channels ◽  
Cortical Collecting Duct ◽  
Whole Cell ◽  
Cell Current ◽  
Whole Cell Current

In the present study we used whole-cell patch clamp recordings to investigate swelling-activated Cl-currents (ICl-swell) in M-1 mouse cortical collecting duct (CCD) cells. Hypotonic cell swelling reversibly increased the whole-cell Cl- conductance by about 30-fold. The I-V relationship was outwardly-rectifying and ICl-swell displayed a characteristic voltage-dependence with relatively fast inactivation upon large depolarizing and slow activation upon hyperpolarizing voltage steps. Reversal potential measurements revealed a selectivity sequence SCN- > I- > Br- > Cl- > > gluconate. ICl-swell was inhibited by tamoxifen, NPPB (5-nitro-2(3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), flufenamic acid, niflumic acid, and glibenclamide, in descending order of potency. Extracellular cAMP had no significant effect. ICl-swell was Ca2+ independent, but current activation depended on the presence of a high-energy gamma-phosphate group from intracellular ATP or ATP gamma S. Moreover, it depended on the presence of intracellular Mg2+ and was inhibited by staurosporine, which indicates that a phosphorylation step is involved in channel activation. Increasing the cytosolic Ca2+ concentration by using ionomycin stimulated Cl- currents with a voltage dependence different from that of ICl-swell. Analysis of whole-cell current records during early onset of ICl-swell and during final recovery revealed discontinuous step-like changes of the whole-cell current level which were not observed under nonswelling conditions. A single-channel I-V curve constructed using the smallest resolvable current transitions detected at various holding potentials and revealed a slope conductance of 55, 15, and 8 pS at +120, 0, and -120 mV, respectively. The larger current steps observed in these recordings had about 2, 3, or 4 times the size of the putative single-channel current amplitude, suggesting a coordinated gating of several individual channels or channel subunits. In conclusion we have functionally characterized ICl-swell in M-1 CCD cells and have identified the underlying single channels in whole-cell current recordings.

scholarly journals RNA sequencing of kidney distal tubule cells reveals multiple mediators of chronic aldosterone action

2018 ◽  
Vol 50 (5) ◽  
pp. 343-354 ◽  
Author(s):  
Søren Brandt Poulsen ◽  
Kavee Limbutara ◽  
Robert A. Fenton ◽  
Trairak Pisitkun ◽  
Birgitte Mønster Christensen
Keyword(s):  
Rna Sequencing ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Cell Types ◽  
Sodium Reabsorption ◽  
Specific Cell ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Mrna Transcriptome

The renal aldosterone-sensitive distal tubule (ASDT) is crucial for sodium reabsorption and blood pressure regulation. The ASDT consists of the late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct. Due to difficulties in isolating epithelial cells from the ASDT in large quantities, few transcriptome studies have been performed on this segment. Moreover, no studies exist on isolated DCT2 and CNT cells (excluding intercalated cells), and the role of aldosterone for regulating the transcriptome of these specific cell types is largely unknown. A mouse model expressing eGFP in DCT2/CNT/initial cortical collecting duct (iCCD) principal cells was exploited to facilitate the isolation of these cells in high number and purity. Combined with deep RNA sequencing technology, a comprehensive catalog of chronic aldosterone-regulated transcripts from enriched DCT2/CNT/iCCD principal cells was generated. There were 257 significantly downregulated and 290 upregulated transcripts in response to aldosterone ( P < 0.05). The RNA sequencing confirmed aldosterone regulation of well-described aldosterone targets including Sgk1 and Tsc22d3. Changes in selected transcripts such as S100a1 and Cldn4 were confirmed by RT-qPCR. The RNA sequencing showed downregulation of Nr3c2 encoding the mineralocorticoid receptor (MR), and cell line experiments showed a parallel decrease in MR protein. Furthermore, a large number of transcripts encoding transcription factors were downregulated. An extensive mRNA transcriptome reconstruction of an enriched CNT/iCCD principal cell population was also generated. The results provided a comprehensive database of aldosterone-regulated transcripts in the ASDT, allowing development of novel hypotheses for the action of aldosterone.

scholarly journals The mechanosensitive BKα/β1 channel localizes to cilia of principal cells in rabbit cortical collecting duct (CCD)

2017 ◽  
Vol 312 (1) ◽  
pp. F143-F156 ◽  
Author(s):  
Rolando Carrisoza-Gaytán ◽  
Lijun Wang ◽  
Carlos Schreck ◽  
Thomas R. Kleyman ◽  
Wen-Hui Wang ◽  
...  
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
High Amplitude ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
High K ◽  
Dependent Flow

Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca2+- and/or stretch-dependent flow-induced K+ secretion (FIKS) and contributes to K+ adaptation in response to dietary K+ loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K+ secretory processes. In support of a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore-forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high-K+ diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca2+ concentration, albeit of nonciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K+-fed rabbits with channel subunit-specific antibodies revealed colocalization of immunodetectable BKα- and β1-subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low-K+ vs. high-K+ diet. Single-channel recordings of cilia revealed K+ channels with conductance and kinetics typical of the BK channel. The observations that 1) FIKS was preserved but 2) the high-amplitude Ca2+ peak elicited by flow was reduced in microperfused CCDs subject to pharmacological deciliation suggest that cilia BK channels do not contribute to K+ secretion in this segment, but that cilia serve as modulators of cell signaling.

Kir4.1/Kir5.1 channel forms the major K+ channel in the basolateral membrane of mouse renal collecting duct principal cells

2008 ◽  
Vol 294 (6) ◽  
pp. F1398-F1407 ◽  
Author(s):  
Sahran Lachheb ◽  
Françoise Cluzeaud ◽  
Marcelle Bens ◽  
Mathieu Genete ◽  
Hiroshi Hibino ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Hill Coefficient ◽  
Specific Marker ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Principal Cells ◽  
Inside Out

K+ channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K+ channels with conductances of ∼75, 40, and 20 pS were observed, but the K+ channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg2+-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg2+ had no influence on the inward conductance ( Gin = 35 pS) but reduced outward conductance ( Gout) to 13 pS, yielding a Gin/ Gout of 3.2. The polycation spermine (6 × 10−7 M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pHi): a sigmoid relationship between pHi and channel normalized current ( NPo) was observed with a p K of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K+ (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K+ had no influence on the properties of the intermediate-conductance channel, but a Na+-depleted diet increased its open probability by ∼25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K+ conductance in the basolateral membrane of mouse CCD principal cells.

scholarly journals Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct

2008 ◽  
Vol 295 (4) ◽  
pp. F1063-F1070 ◽  
Author(s):  
Vladislav Bugaj ◽  
Oleh Pochynyuk ◽  
Elena Mironova ◽  
Alain Vandewalle ◽  
Jorge L. Medina ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Na Channel ◽  
Open Probability ◽  
Endothelin 1 ◽  
Principal Cells ◽  
Patch Clamp Electrophysiology ◽  
Src Family Tyrosine Kinases

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na+ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETB but not ETA receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.

scholarly journals Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

1991 ◽  
Vol 98 (1) ◽  
pp. 131-161 ◽  
Author(s):  
J F García-Díaz
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Reversal Potential ◽  
Outward Current ◽  
Open Probability ◽  
Whole Cell ◽  
Open Time ◽  
Single Channel Currents ◽  
External K

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

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