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.

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

1999 ◽  
Vol 277 (5) ◽  
pp. C913-C925 ◽  
Author(s):  
Thierry van den Abbeele ◽  
Jacques Teulon ◽  
Patrice Tran Ba Huy
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Guinea Pig Cochlea ◽  
Voltage Dependent ◽  
Inside Out

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K+channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K+ channels. A Ca2+-activated K+ channel with a high conductance (220 pS, P K/ P Na= 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca2+concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K+ channel had a low conductance and a small outward rectification in symmetrical K+ conditions (10 pS for inward currents and 20 pS for outward currents, P K/ P Na= 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 μM acetylcholine. It was not sensitive to internal Ca2+, was inhibited by 4-aminopyridine, was activated by depolarization above −30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

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 Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct.

1991 ◽  
Vol 98 (1) ◽  
pp. 35-61 ◽  
Author(s):  
W Wang ◽  
G Giebisch
Keyword(s):  
Potassium Channel ◽  
Collecting Duct ◽  
Hill Coefficient ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Open Probability ◽  
High Concentrations ◽  
The Hill ◽  
Inside Out ◽  
Small Conductance

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.

Chloride channels in the apical membrane of cortical collecting duct cells

1990 ◽  
Vol 258 (2) ◽  
pp. F273-F280 ◽  
Author(s):  
D. B. Light ◽  
E. M. Schwiebert ◽  
G. Fejes-Toth ◽  
A. Naray-Fejes-Toth ◽  
K. H. Karlson ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Rabbit Kidney ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Permeability Ratio ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cells In Culture ◽  
Inside Out

Ion channels in the apical membrane of cortical collecting duct (CCD) cells in culture were studied by the patch-clamp technique. CCD cells from rabbit kidney were isolated by solid-phase immunoadsorption with a monoclonal antibody. The majority of CCD cells (93%) had phenotypic characteristics similar to intercalated cells (ICC). Although Cl- channels were present in the apical membrane of the ICC cells, they were rarely active in cell-attached patches; however, channels were activated after patch excision. In inside-out patches, the channels exhibited rapid flickering, substrates, and large unitary currents. The single-channel conductance was 303 pS, the Cl(-)-to-Na+ permeability ratio was 10:1 and the Cl(-)-to-HCO3- permeability ratio was 1.5:1. The channel was inhibited by the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Although a reduction in the cytoplasmic Ca2+ concentration inhibited channel activity in both inside-out patches and cell-attached patches, alterations of Ca2+ within the physiological range did not change the channel open probability. Finally, changing the cytoplasmic pH (6.5 to 8.0) did not alter the open probability. Thus a large conductance anion channel is present in the apical membrane of CCD cells in culture. This channel may be involved in cell volume regulation or in Cl- and HCO3- secretion.

Properties of an inwardly rectifying K+ channel in the basolateral membrane of mouse TAL

2002 ◽  
Vol 282 (5) ◽  
pp. F866-F876 ◽  
Author(s):  
Marc Paulais ◽  
Stéphane Lourdel ◽  
Jacques Teulon
Keyword(s):  
Inhibitory Concentration ◽  
Basolateral Membrane ◽  
Hill Coefficient ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Effective Valence ◽  
Inwardly Rectifying ◽  
Inside Out

We investigated the properties of K+ channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K+ channel (K+-to-Na+permeability ratio ∼22), having an inward conductance ( G in) of 44 pS and an outward conductance ( G out) of ∼10 pS ( G in/ G out ∼ 4). Channel activity ( NP o) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg2+-free medium, the channel had a G in of 50 pS and was weakly inwardly rectifying ( G in/ G out ∼ 1). Cytosolic Mg2+ reduced G out, yielding a G in/ G out of 3.8 at 1.3 mM Mg2+. Internal Na+ also yielded a G in/ G out of 1.6 at 20 mM Na+. Spermine reduced NP o on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration ( K i) = 0.2 μM] was much greater than at hyperpolarizing voltages ( K i = 26 μM). Half-inactivation by 0.5 μM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NP o of inside-out membrane patches was observed, with a p K of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NP o of cell-attached patches. This channel is probably a major component of K+ conductance in the CTAL basolateral membrane.

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.

Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture

1992 ◽  
Vol 263 (2) ◽  
pp. F243-F250 ◽  
Author(s):  
P. Dietl ◽  
B. A. Stanton
Keyword(s):  
Chloride Channels ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Cl Channels ◽  
Inside Out ◽  
Cl Conductance

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.

Insulin increases the activity of mesangial BK channels through MAPK signaling

2008 ◽  
Vol 294 (6) ◽  
pp. F1465-F1472 ◽  
Author(s):  
Ruth M. Foutz ◽  
P. Richard Grimm ◽  
Steven C. Sansom
Keyword(s):  
Plasma Membrane ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Early Stage ◽  
Bk Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
High Insulin

Glomerular hyperfiltration and mesangial expansion have been described in mouse models of a hyperinsulinemic early stage of type 2 diabetes mellitus (DM). Large-conductance Ca2+-activated K+ channels (BK) have been linked to relaxation of human mesangial cells (MC) and may contribute to MC expansion and hyperfiltration. We hypothesized that high insulin levels increase BK activity in MC by increasing the number and/or open probability ( Po) of BK in the plasma membrane. With the use of the patch-clamp technique, BK activity was analyzed in cultured MC exposed to normal insulin (1 nM) and high insulin (100 nM) for a 48-h period. The mean Po and the percentage of patches (cell attached) with detected BK increased by 100% in the insulin-treated cells. Real-time PCR revealed that insulin increased mRNA of BK-α. Western blot revealed an insulin-stimulated increase in BK-α from both total cellular and plasma membrane protein fractions. The mitogen-activated protein kinase (MAPK) inhibitors PD-098059 and U-0126 attenuated the insulin-induced increase in BK-α expression. PD-098059 inhibited insulin-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 in MC. An insulin-stimulated increase also was found for total cellular BK-β1, the accessory subunit of BK in MC. A similar increase in BK-α mRNA and protein was evoked by an insulin-like growth factor I analog. Glomeruli, isolated from hyperinsulinemic early stage type 2 DM mice, exhibited increased BK-α mRNA by real-time PCR and protein by immunohistochemical staining and Western blot. These results indicate that insulin activates BK in the plasma membrane of MC and stimulates, via MAPK, an increase in cellular and plasma membrane BK-α.

cGMP-activating peptides do not regulate electrogenic electrolyte transport in principal cells of rat CCD

1996 ◽  
Vol 271 (6) ◽  
pp. F1158-F1165 ◽  
Author(s):  
E. Schlatter ◽  
R. Cermak ◽  
W. G. Forssmann ◽  
J. R. Hirsch ◽  
R. Kleta ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Electrolyte Transport ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Principal Cells ◽  
Intracellular Cgmp ◽  
Whole Cell Patch Clamp

K+ channels in the basolateral membrane of rat cortical collecting duct (CCD) are regulated by a cGMP-dependent protein kinase (J. Hirsch and E. Schlatter. Pfluegers Arch. 429: 338-344, 1995). Conflicting data exist on the effects of cGMP-activating agonists on Na+ transport in these cells. Thus we tested members of the family of peptides that increase intracellular cGMP [cardiodilatin/atrial natriuretic peptide (CDD/ANP), brain natriuretic peptide, C-type natriuretic peptide, urodilatin, guanylin, and uroguanylin], as well as bradykinin +/- CDD/ANP on membrane voltages (Vm) of principal cells of isolated rat CCD using the slow whole cell patch-clamp technique (E. Schlatter, U. Frobe, and R. Greger. Pfluegers Arch. 421: 381-387, 1992). None of the agonists tested changed Vm significantly. There was also no effect of dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) on AVP-dependent lumen-to-bath Na+ flux, transepithelial voltage, or osmotic water permeability in isolated perfused rat CCD. Finally, CDD/ANP increased intracellular cGMP only in glomeruli but not in CCD. Thus the findings provide no evidence for control of electrogenic electrolyte transport by these natriuretic peptides in principal cells of rat CCD, and the agonist that physiologically regulates the cGMP-dependent K+ channels remains to be identified.

scholarly journals Adenosine inhibits the basolateral Cl− ClC-K2/b channel in collecting duct intercalated cells

2020 ◽  
Vol 318 (4) ◽  
pp. F870-F877
Author(s):  
Oleg Zaika ◽  
Viktor N. Tomilin ◽  
Oleh Pochynyuk
Keyword(s):  
Salt Intake ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
Intercalated Cells ◽  
Dietary Salt ◽  
Open Probability ◽  
Salt Loading ◽  
Principal Cells ◽  
High Salt Intake

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1 receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+ conductance mediated by Kir4.1/5.1 channels to mediate K+ recycling and to set up a favorable driving force for Na+/K+ exchange ( 47 ). Intercalated cells express the Cl− ClC-K2/b channel mediating transcellular Cl− reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl− reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

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