Volume-sensitive basolateral K+ channels in HT-29/B6 cells: block by lidocaine, quinidine, NPPB, and Ba2+ (original) (raw)
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Biochemical and Biophysical Research Communications, 2001
The rat primary cultured-airway monolayer has been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane. Inwardly rectifying K ؉ currents were characterized across the basolateral membrane in symmetrical HCO 3 ؊-free high K ؉ Ringer's solution (125 mM) in this study. The potency of K ؉ channel inhibitors against K ؉ conductance was Ba 2؉ (IC 50 ؍ 5 M) > Cs ؉ (IC 50 ؍ 2 mM) ӷ glybenclamide (IC 50 > 5 mM) ӷ TEA (IC 50 ӷ 100 mM). The application of basolateral Cs ؉ changed K ؉ conductance into an oscillating current, and its frequency (holding voltage ؍ ؊100 mV) increased with increase in concentration of basolateral Cs ؉ (0.05-5 mM) and in degree of hyperpolarization. Addition of basolateral Cs ؉ blocked inward current strongly at ؊100 mV and hardly at all at ؊60 mV, giving a sharp curvature to the I-V relation of the IRK current. RT-PCR, Western blotting, and immunohistochemical analyses showed that Kir2.1 might be present in basolateral membrane of tracheal epithelia and plasma membrane of pulmonary alveolar cells.
AJP: Cell Physiology, 2004
Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca(2+) dependent. In nominally Ca(2+)-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial -79 mV to a steady state of -54 mV reached at 18% osmolarity reduction [hyposmolarity of -18% (H-18%)]. Recordings of Cl(-) and K(+) currents showed activation at H-3% of an outwardly rectifying Cl(-) current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K(+) current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K(+) channel blockers. With 200 nM Ca(2+) in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from -77 to -63 mV. A K(+) current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K(+) channels in response to GOR in the same cell type, activated by Ca(2+) and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl(-) and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.
The Journal of Membrane Biology, 1994
We describe the activation of a K + current and inhibition of a C1-current by a cyanoguanidine activator of ATP-sensitive K + channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KAT P was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 ~tM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 ~tM). The U83757induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlylammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 ~tM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where E K =-82 mV and Ecl = 0 mV, the reversal potential of the U83757-sensitive current was approximately-70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a C1-channel blocker as well as a K +chan-Correspondence to: DJ. Nelson nel opener. In experiments in which C1-current activation was specifically brought about by cellular swelling and performed in solutions where C1-was the major permeant ion, U83757 (300 nM tO 300 gM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K+-selective current which is sensitive to KAy P channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K + channel openers in vascular smooth muscle may be due to both the inhibition of C1currents as well as the activation of a K+-selective current.
Pfl�gers Archiv European Journal of Physiology, 1999
Secretion of Clrequires the presence of a K + conductance to hyperpolarize the cell, and to provide the driving force for Clexit via luminal Clchannels. In the exocrine pancreas Clsecretion is mediated by an increase in cytosolic Ca 2+ ([Ca 2+ ] i). Two types of Ca 2+-activated K + channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca 2+-activated K + channels in rat pancreatic acini. Here we examine the basolateral K + conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 µmol/l) to the bath led to the activation of very small conductance K + channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1±0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f c) of 30.3±3.5 Hz (n=19), consistent with channel activity in these patches. The estimated singlechannel conductance was 1.5±0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K + in the bath solution. Under these conditions f c was 47.6±11.9 Hz (estimated single-channel conductance 1.1±0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E K + when Na + in the bath was replaced by K +. Channel activity in cellexcised patches was slightly reduced by 10 mmol/l Ba 2+ (23.6±2.1% of the total outward current) and was completely absent when K + in the bath was replaced by Na +. Reduction of the [Ca 2+ ] i from 1 mmol/l to 1 µmol/l in cell-excised experiments decreased the current by 52.3±12.3% (n=5). Expression of K V LQT1, one of the possible candidates for a small-conductance K + channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K V LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K + channels in rat pancreatic acinar cells, most likely via an increase in [Ca 2+ ] i .
Millimolar amiloride concentrations block K conductance in proximal tubular cells
British Journal of Pharmacology, 1992
Amiloride, applied at millimolar concentrations, results in the blockade of K+ conductance in amphibian proximal convoluted cells (PCT), fused into giant cells. 2 Amiloride results directly in a blockade of K+ conductance that is not related to inhibition of the Na+-H+ antiport, which would lower intracellular pH, adversely affecting K+ conductance. On the contrary, high amiloride concentrations promote entry of this lipophilic base in the cell, leading to higher cell pH. 3 Under voltage clamp conditions, control vs. amiloride, current-voltage curves from PCT fused giant cells intersect at-86.2 ± 3.4 mV, a value close to the equilibrium potential for potassium. 4 Hexamethylene amiloride, 10-5 M, irreversibly depolarizes the membrane potential. 5 Barium decreased by 50% the initial slope of realkalinization, following removal of a solution containing NH4Cl, as did amiloride. In addition, these blockers reduced membrane conductance by 40%, suggesting that a fraction of the amiloride-suppressible NH4' efflux may be conductive. 6 Amiloride does not directly inhibit the Na+-K+, ATPase in our preparation, contrary to the prevalent-belief. 7 In vivo studies show that amiloride interferes with an apical K+ conductance but it does not alter basolateral K+ conductance.
Resting and osmotically induced basolateral K conductances in turtle colon
The Journal of General Physiology, 1986
A B STR A CT Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986,Journal of General Physiology, 88 :237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCI) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling . In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidinesensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a
Basolateral potassium membrane permeability of A6 cells and cell volume regulation
The Journal of Membrane Biology, 1994
The K + permeabilities (86Rb(K) transport) of the basolateral membranes (Jb K) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation. Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca (max < 1 min) and cell swelling (max at 3-5 rain) followed by a regulatory volume decrease (5-30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the S6Rb(K) transport and the SCC were partially blocked by Ba 2+, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3-6 rain) stimulation of the 86Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca2+-free media or quinidine addition did not affect the initial osmotic stimulation of Jb K but prevented its "secondary regulation," whereas TEA, glibenclamide and DIDS completely blocked the initial Jb K increase. Under hypo-osmotic conditions, the initial Jb K increase was enhanced by the presence of 1 mM of barium and delayed with higher concentrations (5 ram). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media. We propose that a TEA-and glibenclamide-sensitive but quinidine-insensitive increase in K + permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K + permeability mediated by the observed calcium rise.
Apical and basolateral conductance in cultured A6 cells
Pfl�gers Archiv European Journal of Physiology, 1991
Confluent monolayers of the cultured renal distal tubule cell line (A6) were impaled with microelectrodes under short-circuit conditions. Specific membrane conductances were calculated from equivalent circuit equations. Transport properties of the apical and basolateral membranes were investigated during control conditions and short-term increases in basolateral potassium concentration [K +] from 2.5 to 20 mmol/1, with or without 0.5 mmol/1 Ba 2+ at the basolateral side. As in most other epithelia, the apical membrane represents the major resistive barrier. Transcellular, apical and basolateral membrane conductances (go, go and gi respectively), obtained from 22 acceptable microelectrode studies, averaged 61, 80 and 292 gS/cm 2, respectively. There was a highly significant correlation between short-circuit current (Isc) and go, whereas gi was unrelated to I~c. The /~c, which averaged 4.1 gA/cm 2, was almost completely blocked by amiloride. This was associated with fast hyperpolarization; the intracellular potential (V~o) increased from -69 to -83 mV and the fractional apical resistance rose to nearly 100%. Using the values of V~ during amiloride at normal and high [K+], an apparent transference number for K + at the basolateral membrane of 0.72 can be calculated. This value corresponds with the decrease in g~ to about 25% of the control values after blocking the K + channels with Ba 2 +. The nature of the remaining conductance is presently unclear. The cellular current decreased during high [K +] and Ba 2+, in part resulting from reduction of the electrochemical gradient for apical Na + uptake due to the depolarization. In addition, go decreased to less than 40%, which is considerably lower than predicted by the constant-field equation; this might indicate voltage sensitivity of the apical Na + permeability.
The Journal of Physiology, 2002
Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K + channels involved in basal, cAMPand Ca 2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I sc). The non-specific K + channel inhibitor Ba 2+ inhibited the basal I sc by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K + conductance is required for maintenance of basal anion secretion. Using the K + channel blockers clofilium and clotrimazole, we found basal I sc to be sensitive to clofilium, with a small clotrimazolesensitive component. By stimulating the cAMP and Ca 2+ pathways, we determined that cAMPstimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca 2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K + channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K + conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I sc. Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K + channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca 2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K + channel types, possibly involving one or more class of KCNQ1-containing channel complexes.
Basolateral membrane conductance in A6 cells: effect of high sodium transport rate
Pflugers Archiv European Journal of Physiology, 1992
Conductance of apical and basolateral membranes in short-circuited cultured renal distal cells (A6) was determined using microelectrodes. Epithelia were pre-incubated with 0.1 ~mol/1 dexamethasone in the presence of 4 ~tmol/1 amiloride to prevent increase in apical Na + entry. Omission of amiloride increased the I~c from 5.7 to 27.6 gA/cm 2 due to the rise in apical membrane conductance from 21 to 595 gS/cm 2. Apical fractional resistance decreased from 0.89 to 0.40 and cells depolarized from -52 to -4 mV. Basolateral membrane conductance, which was 320 ~tS/cm 2 at partially inhibited transport, was not significantly altered during the first 2 min following establishment of high transport activity; it started to increase thereafter reaching a more than threefold higher value of 1324 gS/cm 2 within 12min. The gain cannot be explained by increase in partial K + conductance. Disappearance of the conductance after reduction of basolateral C1-or in the presence of the C1 channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate indicates a C1 conductance, which appears to be activated by depolarization.