Apical membrane potassium channels in frog diluting segment: stimulation by furosemide

1992 ◽  
Vol 262 (4) ◽  
pp. F606-F614 ◽  
Author(s):  
A. M. Hurst ◽  
M. Hunter
Keyword(s):  
Potassium Channels ◽  
Apical Membrane ◽  
Inward Rectification ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
High Potassium ◽  
Diluting Segment ◽  
Pipette Solution ◽  
Significant Change

The patch-clamp technique was used to study the activity of apical membrane potassium channels in frog isolated everted diluting segments, and the effect of transport inhibitors on channel activity was assessed. In cell-attached patches with a high-potassium pipette solution and Ringer in the bath the channels show inward rectification (inward conductance, 25.1 pS; outward conductance, 10.5 pS). The channel is selective for potassium over sodium and is voltage dependent with depolarization increasing channel open probability (Po). Furosemide increased channel activity, which resulted exclusively from a significant increase in the number (N) of channels in the patch (control, 2.3 +/- 0.3, n = 8; furosemide, 4.0 +/- 0.4, n = 14) without any significant change in Po. Amiloride blocked the stimulatory effect of furosemide by reducing N to 1.4 +/- 0.6 (n = 6), and amiloride alone also reduced N with no significant change in Po. This suggests that the increase in N in response to furosemide may be secondary to a rise in intracellular pH mediated by activation of the apical Na-H exchanger.

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.

Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule

1990 ◽  
Vol 259 (3) ◽  
pp. F494-F502 ◽  
Author(s):  
W. H. Wang ◽  
A. Schwab ◽  
G. Giebisch
Keyword(s):  
Intracellular Ph ◽  
Apical Membrane ◽  
K Channel ◽  
Selectivity Ratio ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Mean Lifetime ◽  
Collecting Tubules ◽  
Inside Out

We used the patch-clamp technique to study the activity and regulation of single potassium channels in the apical membrane of isolated cortical collecting tubules (CCT) of rat kidney. With 140 mM K+ in the pipette the inward conductance of the channel in cell-attached patches at 37 degrees C was 35 pS (n = 106, NaCl-Ringer or 70 mM KCl and 70 mM NaCl in the bath), and the outward conductance was 15 pS (n = 15, 70 mM NaCl + 70 mM KCl in the bath). Mean open probability (Po) of the channel is voltage independent and 0.96 (n = 106). The channel displayed one open state with a mean lifetime of 18.6 ms and one closed state with a mean lifetime of 0.7 ms (n = 20). Selectivity ratio between K+ and Na+ is 20 (n = 5). High-potassium diet increased channel incidence from control 32% (53 patches with channel from 165 patches) to 64% (53 patches with channels from 83 patches). The channel could be blocked by 1 mM Ba2+ (n = 7, Ba2+ in the pipette); however, 5 mM tetraethylammonium (n = 9, TEA in the pipette) did not block the channel activity. The channel was very sensitive to intracellular pH (n = 6). Changing bath pH facing cytoplasmic side of inside-out patches from 7.4 to 6.9 reversibly reduced Po from 0.9 to 0.1. Addition of 1 mM ATP (n = 7) to bath almost completely inhibited channel activity in inside-out patches. This ATP-induced inhibition was fully reversible and was found to be dependent on the ratio of ATP to ADP, since adding 0.5 mM ADP to bath solution relieved the ATP-induced blockade. Results indicate that intracellular pH, concentration of ATP, and ratio of ATP to ADP are important regulators of potassium channel activity in the apical membrane of rat CCT, and the properties of the channel make it a strong candidate for K+ secretion in this nephron segment.

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

Dietary K intake regulates the response of apical K channels to adenosine in the thick ascending limb

2004 ◽  
Vol 287 (5) ◽  
pp. F954-F959 ◽  
Author(s):  
Dimin Li ◽  
Yuan Wei ◽  
Wen-Hui Wang
Keyword(s):  
Adenosine Receptor ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cgs 21680 ◽  
Adenosine Receptor Agonist ◽  
Dependent Pathway ◽  
Adenosine Analog

We used the patch-clamp technique to study the effect of adenosine on the apical 70-pS K channel in the thick ascending limb (TAL) of the rat kidney. Application of 1 μM cyclohexyladenosine (CHA), an adenosine analog, stimulated apical 70-pS K channel activity and increased the product of channel open probability and channel number ( NPo) from 0.34 to 0.7. Also, addition of CGS-21680, a specific A2a adenosine receptor agonist, mimicked the effect of CHA and increased NPo from 0.33 to 0.77. The stimulatory effect of CHA and CGS-21680 was completely blocked by H89, an inhibitor of protein kinase A (PKA), or by inhibition of adenylate cyclase with SQ-22536. This suggests that the stimulatory effect of adenosine analogs is mediated by a PKA-dependent pathway. The effect of adenosine analog was almost absent in the TAL from rats on a K-deficient (KD) diet for 7 days. Application of DDMS, an agent that inhibits cytochrome P-450 hydrolase, not only significantly increased the activity of the 70-pS K channel but also restored the stimulatory effect of CHA on the 70-pS K channel in the TAL from rats on a KD diet. Also, the effect of CHA was absent in the presence of 20-HETE. Inhibition of PKA blocked the stimulatory effect of CHA on the apical 70-pS K channel in the presence of DDMS in the TAL from rats on a KD diet. We conclude that stimulation of adenosine receptor increases the apical 70-pS K channel activity via a PKA-dependent pathway and that the effect of adenosine on the apical 70-pS K channel is suppressed by low-K intake. Moreover, the diminished response to adenosine is the result of increase in 20-HETE formation, which inhibits the cAMP-dependent pathway in the TAL from rats on a KD diet.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1036-C1044 ◽  
Author(s):  
Z. Fan ◽  
Y. Tokuyama ◽  
J. C. Makielski
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Activation Effect ◽  
Insulin Secreting Cells ◽  
Single Channel Currents

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

Chloride channels in primary cultures of seawater fish (Dicentrarchus labrax) gill

1997 ◽  
Vol 273 (3) ◽  
pp. C874-C882 ◽  
Author(s):  
C. Duranton ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Chloride Channels ◽  
Primary Cultures ◽  
Ion Homeostasis ◽  
Dicentrarchus Labrax ◽  
Disulfonic Acid ◽  
Inward Rectification ◽  
Open Probability ◽  
Pipette Solution ◽  
Cl Channel ◽  
Respiratory Cells

Patch-clamp experiments were undertaken on primary cultures of respiratory cells originating from sea bass (Dicentrarchus labrax) gills. A small-conductance Cl- channel of 8 pS was characterized in cell-attached configuration with 140 mM N-methyl-D-glucamine-Cl in the pipette and bath solutions. No activity was recorded below a membrane holding potential of +20 mV (-Vp, referenced to the pipette solution), and the channel showed an inward rectification. In the inside-out configuration the Cl- channel was active at all membrane holding potentials. Its open probability strongly increased with membrane depolarization. The channel activity could be increased by the application of protein kinase A+ATP. This channel was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, diphenylamino-2-carboxylic acid, and I- and was insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of Cl- channels in the apical membrane of respiratory cells provides additional evidence for an important role of this cell type in the control of ion homeostasis of seawater fish.

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K+ channels

2000 ◽  
Vol 279 (5) ◽  
pp. R1647-R1658 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Potassium Channels ◽  
Primary Cultures ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Ionic Substitution ◽  
Gill Cells

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl− channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K+ channel of 123 ± 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K+ over Na+ and Ca2+. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 μM), and gadolinium (500 μM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K+ channel activity is consistent with the hypothesis that stretch-activated K+ channels could mediate an increase in K+ conductance during cell swelling.

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 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.

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