Apical potassium channels in the rat connecting tubule

2004 ◽  
Vol 287 (5) ◽  
pp. F1030-F1037 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Membrane Voltage ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Connecting Tubule ◽  
Channel Type

Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the “SK”-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 ± 24 pA/cell) or 7 days (385 ± 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen.

scholarly journals Apical membrane of native OMCDi cells has nonselective cation channels

2001 ◽  
Vol 281 (1) ◽  
pp. F48-F55 ◽  
Author(s):  
Shen-Ling Xia ◽  
Seung-Hyun Noh ◽  
Jill W. Verlander ◽  
Craig H. Gelband ◽  
Charles S. Wingo
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Reversal Potential ◽  
Cation Channels ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Potassium Gluconate ◽  
Medullary Collecting Duct

The purpose of this study was to examine cation channel activity in the apical membrane of the outer medullary collecting duct of the inner stripe (OMCDi) using the patch-clamp technique. In freshly isolated and lumen-opened rabbit OMCDi, we have observed a single channel conductance of 23.3 ± 0.6 pS ( n = 17) in cell-attached (c/a) patches with high KCl in the bath and in the pipette at room temperature. Channel open probability varied among patches from 0.06 ± 0.01 at −60 mV ( n = 5) to 0.31 ± 0.04 at 60 mV ( n = 6) and consistently increased upon membrane depolarization. In inside-out (i/o) patches with symmetrical KCl solutions, the channel conductance (22.8 ± 0.8 pS; n = 10) was similar as in the c/a configuration. Substitution of the majority of Cl− with gluconate from KCl solution in the pipette and bath did not significantly alter reversal potential ( E rev) or the channel conductance (19.7 ± 1.1 pS in asymmetrical potassium gluconate, n = 4; 21.4 ± 0.5 pS in symmetrical potassium gluconate, n = 3). Experiments with 10-fold lower KCl concentration in bath solution in i/o patches shifted E rev to near the E rev of K+. The estimated permeability of K+ vs. Cl− was over 10, and the conductance was 13.4 ± 0.1 pS ( n = 3). The channel did not discriminate between K+ and Na+, as evidenced by a lack of a shift in the E rev with different K+ and Na+ concentration solutions in i/o patches ( n = 3). The current studies demonstrate the presence of cation channels in the apical membrane of native OMCDicells that could participate in K+ secretion or Na+ absorption.

Two types of K+ channel in thick ascending limb of rat kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
W. H. Wang
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inside Out

We have used the patch-clamp technique to study the apical K+ channels in the thick ascending limb (TAL) of the rat kidney. Two types of K+ channels, a low-conductance and an intermediate-conductance K+ channel, were identified in both cell-attached and inside-out patches. We confirmed the previously reported intermediate-conductance K+ channel (72 pS), which is inhibited by millimolar cell ATP, acidic pH, Ba2+, and quinidine (4). We now report a second K+ channel in apical membrane of the TAL. The slope conductance of this low-conductance K+ channel is 30 pS, and its open probability is 0.80 in cell-attached patches. This channel is not voltage dependent, and application of 2 mM ATP in the bath inhibits channel activity in inside-out patches. In addition, 250 microM glyburide, an ATP-sensitive K+ channel inhibitor, blocks channel activity, whereas the same concentration of glyburide has no inhibitory effect on the 72-pS K+ channel. Channel activity of the 30-pS K+ channel decreases rapidly upon excision of patches (channel run down). Application of 0.1 mM ATP and the catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) restores channel activity. Furthermore, addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP or 50-100 pM vasopressin in the cell-attached patches increases channel activity. In conclusion, two types of K+ channels are present in the apical membrane of TAL of rat kidney, and PKA plays an important role in modulation of the low-conductance K+ channel activity.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

scholarly journals Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

1990 ◽  
Vol 95 (5) ◽  
pp. 791-818 ◽  
Author(s):  
Y Segal ◽  
L Reuss
Keyword(s):  
Patch Clamp ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Depolarization ◽  
Membrane Voltage ◽  
K Channel ◽  
Gallbladder Epithelium ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Necturus Gallbladder

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

scholarly journals Decrease in dietary K intake stimulates the generation of superoxide anions in the kidney and inhibits K secretory channels in the CCD

2010 ◽  
Vol 298 (6) ◽  
pp. F1515-F1522 ◽  
Author(s):  
Zhi-Jian Wang ◽  
Peng Sun ◽  
WenMing Xing ◽  
ChunYang Pan ◽  
Dao-Hong Lin ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Sk Channels ◽  
Superoxide Anions ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Normal Plasma ◽  
K Concentration ◽  
Rats And Mice

We previously demonstrated that K depletion inhibited ROMK-like small-conductance K channels (SK) in the cortical collecting duct (CCD) and that the effect was mediated by superoxide anions that stimulated Src family protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) ( 51 ). However, because animals on a K-deficient diet had a severe hypokalemia, superoxide-dependent signaling may not regulate ROMK channels under physiological conditions with a normal plasma K concentration. In the present study, we used the patch-clamp technique and Western blot to examine the effect of a moderate K restriction on ROMK-like SK channels and the role of PTK and MAPK in regulating apical K channels in the CCD of animals on a low-K diet (LK; 0.1% K). Rats and mice fed a LK diet for 7 days had a normal plasma K concentration. However, a LK intake increased the expression of angiotensin II type 1 receptor in the kidney. Moreover, patch-clamp experiments demonstrated that LK intake decreased the probability finding SK channels and channel activity defined by NPo (a product of channel number and open probability) in the CCD of both rat and mouse kidneys. Also, LK intake significantly stimulated the production of superoxide anions in the renal cortex and outer medulla in both rats and mice and increased superoxide level in the rat CCD. Moreover, LK intake augments the phosphorylation of p38 and ERK MAPK, the expression of c-Src and tyrosine phosphorylation of ROMK channels. However, treatment of animals with tempol abolished the effect of LK intake on MAPK and c-Src and increased ROMK channel activity in comparing with those of nontreated rats on a LK diet. Inhibiting p38 and ERK with SB202190 and PD98059 significantly stimulated SK in the CCD in rats on a LK diet. In addition, inhibition of PTK with herbimycin A activated SK channels in the CCD from rats on a LK diet. We conclude that LK intake stimulates the generation of superoxide anion and related products and that MAPK and Src family PTK play a physiological role in inhibiting apical K channels in the principal cells in response to LK intake.

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.

Dietary K+ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct

2005 ◽  
Vol 289 (4) ◽  
pp. F922-F932 ◽  
Author(s):  
Fadi Najjar ◽  
Hao Zhou ◽  
Tetsuji Morimoto ◽  
James B. Bruns ◽  
Hai-Sheng Li ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
K Channel ◽  
Ambystoma Tigrinum ◽  
Intercalated Cells ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
K Channels ◽  
Channel Density

The cortical collecting duct (CCD) is a final site for regulation of K+ homeostasis. CCD K+ secretion is determined by the electrochemical gradient and apical permeability to K+. Conducting secretory K+ (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K+ secretion, maxi-K channels appear to participate in flow-stimulated K+ secretion. Chronic dietary K+ loading enhances the CCD K+ secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693–710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K+ increases renal K+ excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107–116, 1998). The purpose of the present study was to test whether K+ adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K+ diet for 10–14 days. Real-time PCR quantitation of message encoding maxi-K α- and β2–4-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals ( P < 0.05); β1-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of α-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in α-intercalated cells in HK kidneys. In summary, K+ adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical α-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.

High baseline ROMK activity in mouse late distal convoluted and early connecting tubule (DCT2/CNT) probably contributes to aldosterone-independent K+ secretion

2021 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Marko Bertog ◽  
Christoph Korbmacher
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Standard Diet ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Connecting Tubule ◽  
Low Potassium ◽  
K Secretion ◽  
Baseline Conditions

The renal outer medullary K+ channel (ROMK) is co-localized with the epithelial Na+ channel (ENaC) in late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone-dependent in late CNT and early CCD (CNT/CCD) but aldosterone-independent in DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone ROMK-mediated K+ secretion mainly occurs in DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in DCT2/CNT than in CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in DCT2/CNT and CNT/CCD microdissected from mice maintained on standard diet. In single-channel recordings from outside-out patches we detected typical ROMK channel activity in both DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive (ΔIami) and tertiapin-sensitive (ΔITPNQ) whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline ΔIami was high in DCT2/CNT (~370 pA) but low in CNT/CCD (~60 pA). Importantly, ΔITPNQ was significantly higher in DCT2/CNT than in CNT/CCD (~810 pA versus ~350 pA). We conclude that high ROMK activity in DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low potassium diet significantly reduced ENaC but not ROMK activity in DCT2/CNT. This suggests that modifying ENaC activity in DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.

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.

PGE2 inhibits apical K channels in the CCD through activation of the MAPK pathway

2007 ◽  
Vol 293 (4) ◽  
pp. F1299-F1307 ◽  
Author(s):  
Yan Jin ◽  
Zhijian Wang ◽  
Yan Zhang ◽  
Baofeng Yang ◽  
Wen-Hui Wang
Keyword(s):  
Mapk Pathway ◽  
Collecting Duct ◽  
Bk Channels ◽  
Mitogen Activated Protein Kinase ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Deficient Diet ◽  
Mapk Phosphorylation ◽  
K Channels ◽  
Ep1 Receptor

We used the patch-clamp technique and Western blot analysis to explore the effect of PGE2 on ROMK-like small-conductance K (SK) channels and Ca2+-activated big-conductance K channels (BK) in the cortical collecting duct (CCD). Application of 10 μM PGE2 inhibited SK and BK channels in the CCD. Moreover, either inhibition of PKC or blocking mitogen-activated protein kinase (MAPK), P38 and ERK, abolished the effect of PGE2 on SK channels in the CCD. The effect of PGE2 on SK channels was completely blocked in the presence of SC-51089, a specific EP1 receptor antagonist, and mimicked by application of sulprostone, an agonist for EP1 and EP3 receptors. To determine whether PGE2 stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with PGE2. Application of PGE2 significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose-response curve of PGE2 effect shows that 1, 5, and 10 μM PGE2 increased the phosphorylation of P38 and ERK by 20–21, 50–80, and 80–100%, respectively. The stimulatory effect of PGE2 on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of PGE2 on MAPK phosphorylation is PKC dependent. Also, the expression of cyclooxygenase II and PGE2 concentration in renal cortex and outer medulla was significantly higher in rats fed a K-deficient diet than those on a normal-K diet. We conclude that PGE2 inhibits SK and BK channels and that there is an effect of PGE2 on SK channels in the CCD through activation of EP1 receptor and MAPK pathways. Also, high concentrations of PGE2 induced by K restriction may be partially responsible for increasing MAPK activity during K restriction.

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