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.

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.

Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F592-F598 ◽  
Author(s):  
W. H. Wang ◽  
A. Cassola ◽  
G. Giebisch
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Inside Out

We have employed the patch-clamp technique to investigate the role of the actin cytoskeleton in the modulation of the low-conductance K+ channel in the apical membrane of the rat cortical collecting duct (CCD). This K+ channel is inactivated by application of cytochalasin B or D, both compounds known to disrupt actin filaments. The effect of both cytochalasins, B and D, was fully reversible in cell-attached patches, but channel activity could not be fully restored in excised membrane patches. The effect of cytochalasins on channel activity was specific and resulted from depolymerization of the actin cytoskeleton, since application of 10 microM chaetoglobosin C, a cytochalasin analogue that does not depolymerize the actin filaments, had no effect on channel activity in inside-out patches. Addition of either actin monomers or of the polymerizing actin filaments in inside-out patches to the cytosolic medium had no effect on channel activity. This suggests that cytochalasin B- or D-induced inactivation of apical K+ channels is not caused by obstruction of the channel pore by actin. We also observed that channel inhibition by cytochalasin B or D could be blocked by pretreatment with 5 microM phalloidin, a compound that stabilizes actin filaments. We conclude that apical K+ channel activity depends critically on the integrity of the actin cytoskeleton.

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.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

1995 ◽  
Vol 268 (4) ◽  
pp. F569-F580 ◽  
Author(s):  
L. C. Stoner ◽  
G. E. Morley
Keyword(s):  
Apical Membrane ◽  
Physiological Role ◽  
Reversal Potential ◽  
K Channel ◽  
Apical Surface ◽  
Open Probability ◽  
Volume Regulatory Decrease ◽  
Collecting Tubules ◽  
High Conductance

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P ' 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

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.

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.

scholarly journals Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

1992 ◽  
Vol 99 (4) ◽  
pp. 591-613 ◽  
Author(s):  
T A Cummings ◽  
S C Kinnamon
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Taste Receptor ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
Inside Out

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

Regulation of single potassium ion channels from apical membrane of rabbit collecting tubule

1986 ◽  
Vol 251 (4) ◽  
pp. F725-F733 ◽  
Author(s):  
M. Hunter ◽  
A. G. Lopes ◽  
E. Boulpaep ◽  
G. Giebisch
Keyword(s):  
Transmembrane Potential ◽  
Apical Membrane ◽  
K Channel ◽  
Potassium Ion Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Barium Concentration ◽  
Collecting Tubule ◽  
Channel Open Time ◽  
Cortical Collecting Tubule

The regulation of K+-channel activity from the apical membrane of the rabbit cortical collecting tubule was studied using the patch-clamp technique. Using inside-out patches, channel open probability was determined as a function of calcium and barium concentration and transmembrane potential. Channel open probability was increased by raising bath (cytoplasmic) calcium concentration, with an apparent Ka of 2.4 microM. Mean channel open time also increased during this maneuver. The channel was reversibly inhibited by barium, applied to the cytoplasmic face, with an apparent Ki of 12 microM. Depolarization of the transmembrane potential increased channel open probability. With 1 mM calcium in the bath solution, the open probability was one-half maximal at -55 mV. It is concluded that this channel is the probable route for transcellular K+ secretion by the cortical collecting tubule and that procedures likely to increase intracellular calcium and/or depolarize the apical membrane will cause an increased potassium secretion.

scholarly journals Regulation of the hyperpolarization-activated K+ channel in the lateral membrane of the cortical collecting duct.

1995 ◽  
Vol 106 (1) ◽  
pp. 25-43 ◽  
Author(s):  
W H Wang
Keyword(s):  
Time Constant ◽  
Voltage Dependence ◽  
Collecting Duct ◽  
K Channel ◽  
Selectivity Ratio ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
Lateral Membrane ◽  
Open Time ◽  
Inside Out

An intermediate-conductance K+ channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K+ channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCl in the pipette and 5 mM KCl, 140 mM NaCl (NaCl Ringer's solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KCl solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/Na+ was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann's equation: Po = 1/[1 + exp(V-V1/2)zF/RT], with z = 1.2 and V1/2 = -40 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K+ channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKAc; 20 U/ml) and 100 microM [corrected] MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKAc increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K+ channel in the basolateral membrane of CCD.

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.

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