Basolateral Potassium Conductance Research Articles

Role of collecting duct principal cell NOS1β in sodium and potassium homeostasis.

The nitric oxide (NO)‐generating enzyme, NO synthase‐1β (NOS1β), is essential for sodium (Na+) homeostasis and blood pressure control. We previously showed that collecting duct principal cell NOS1β is critical for inhibition of the epithelial sodium channel (ENaC) during high Na+ intake. Previous studies on freshly isolated cortical collecting ducts (CCD) demonstrated that exogenous NO promotes basolateral potassium (K+) conductance through basolateral channels, presumably Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16). We, therefore, investigated the effects of NOS1β knockout on Kir4.1/Kir5.1 channel activity. Indeed, in CHO cells overexpressing NOS1β and Kir4.1/Kir5.1, the inhibition of NO signaling decreased channel activity. Male littermate control and principal cell NOS1β knockout mice (CDNOS1KO) on a 7‐day, 4% NaCl diet (HSD) were used to detect changes in basolateral K+ conductance. We previously demonstrated that CDNOS1KO mice have high circulating aldosterone despite a high‐salt diet and appropriately suppressed renin. We observed greater Kir4.1 cortical abundance and significantly greater Kir4.1/Kir5.1 single‐channel activity in the principal cells from CDNOS1KO mice. Moreover, blocking aldosterone action with in vivo spironolactone treatment resulted in lower Kir4.1 abundance and greater plasma K+ in the CDNOS1KO mice compared to controls. Lowering K+ content in the HSD prevented the high aldosterone and greater plasma Na+ of CDNOS1KO mice and normalized Kir4.1 abundance. We conclude that during chronic HSD, lack of NOS1β leads to increased plasma K+, enhanced circulating aldosterone, and activation of ENaC and Kir4.1/Kir5.1 channels. Thus, principal cell NOS1β is required for the regulation of both Na+ and K+ by the kidney.

Deletion of Kir5.1 Impairs Renal Ability to Excrete Potassium during Increased Dietary Potassium Intake.

The basolateral potassium channel in the distal convoluted tubule (DCT), comprising the inwardly rectifying potassium channel Kir4.1/Kir5.1 heterotetramer, plays a key role in mediating the effect of dietary potassium intake on the thiazide-sensitive NaCl cotransporter (NCC). The role of Kir5.1 (encoded by Kcnj16) in mediating effects of dietary potassium intake on the NCC and renal potassium excretion is unknown. We used electrophysiology, renal clearance, and immunoblotting to study Kir4.1 in the DCT and NCC in Kir5.1 knockout (Kcnj16-/- ) and wild-type (Kcnj16+/+ ) mice fed with normal, high, or low potassium diets. We detected a 40-pS and 20-pS potassium channel in the basolateral membrane of the DCT in wild-type and knockout mice, respectively. Compared with wild-type, Kcnj16-/- mice fed a normal potassium diet had higher basolateral potassium conductance, a more negative DCT membrane potential, higher expression of phosphorylated NCC (pNCC) and total NCC (tNCC), and augmented thiazide-induced natriuresis. Neither high- nor low-potassium diets affected the basolateral DCT's potassium conductance and membrane potential in Kcnj16-/- mice. Although high potassium reduced and low potassium increased the expression of pNCC and tNCC in wild-type mice, these effects were absent in Kcnj16-/- mice. High potassium intake inhibited and low intake augmented thiazide-induced natriuresis in wild-type but not in Kcnj16-/- mice. Compared with wild-type, Kcnj16-/- mice with normal potassium intake had slightly lower plasma potassium but were more hyperkalemic with prolonged high potassium intake and more hypokalemic during potassium restriction. Kir5.1 is essential for dietary potassium's effect on NCC and for maintaining potassium homeostasis.

Kir4.1/Kir5.1 in the DCT plays a role in the regulation of renal K+ excretion.

The aim of this mini review is to provide an overview regarding the role of inwardly rectifying potassium channel 4.1 (Kir4.1)/Kir5.1 in regulating renal K+ excretion. Deletion of Kir4.1 in the kidney inhibited thiazide-sensitive NaCl cotransporter (NCC) activity in the distal convoluted tubule (DCT) and slightly suppressed Na-K-2Cl cotransporter (NKCC2) function in the thick ascending limb (TAL). Moreover, increased dietary K+ intake inhibited, whereas decreased dietary K+ intake stimulated, the basolateral potassium channel (a Kir4.1/Kir5.1 heterotetramer) in the DCT. The alteration of basolateral potassium conductance is essential for the effect of dietary K+ intake on NCC because deletion of Kir4.1 in the DCT abolished the effect of dietary K+ intake on NCC. Since potassium intake-mediated regulation of NCC plays a key role in regulating renal K+ excretion and potassium homeostasis, the deletion of Kir4.1 caused severe hypokalemia and metabolic alkalosis under control conditions and even during increased dietary K+ intake. Finally, recent studies have suggested that the angiotensin II type 2 receptor (AT2R) and bradykinin-B2 receptor (BK2R) are involved in mediating the effect of high dietary K+ intake on Kir4.1/Kir5.1 in the DCT.

Potassium intake modulates the thiazide-sensitive sodium-chloride cotransporter (NCC) activity via the Kir4.1 potassium channel

Kir4.1 in the distal convoluted tubule plays a key role in sensing plasma potassium and in modulating the thiazide-sensitive sodium-chloride cotransporter (NCC). Here we tested whether dietary potassium intake modulates Kir4.1 and whether this is essential for mediating the effect of potassium diet on NCC. High potassium intake inhibited the basolateral 40 pS potassium channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule, decreased basolateral potassium conductance, and depolarized the distal convoluted tubule membrane in Kcnj10flox/flox mice, herein referred to as control mice. In contrast, low potassium intake activated Kir4.1, increased potassium currents, and hyperpolarized the distal convoluted tubule membrane. These effects of dietary potassium intake on the basolateral potassium conductance and membrane potential in the distal convoluted tubule were completely absent in inducible kidney-specific Kir4.1 knockout mice. Furthermore, high potassium intake decreased, whereas low potassium intake increased the abundance of NCC expression only in the control but not in kidney-specific Kir4.1 knockout mice. Renal clearance studies demonstrated that low potassium augmented, while high potassium diminished, hydrochlorothiazide-induced natriuresis in control mice. Disruption of Kir4.1 significantly increased basal urinary sodium excretion but it abolished the natriuretic effect of hydrochlorothiazide. Finally, hypokalemia and metabolic alkalosis in kidney-specific Kir4.1 knockout mice were exacerbated by potassium restriction and only partially corrected by a high-potassium diet. Thus, Kir4.1 plays an essential role in mediating the effect of dietary potassium intake on NCC activity and potassium homeostasis.

Dietary K+ and Cl- independently regulate basolateral conductance in principal and intercalated cells of the collecting duct.

The renal collecting duct contains two distinct cell types, principal and intercalated cells, expressing potassium Kir4.1/5.1 (KCNJ10/16) and chloride ClC-K2 (ClC-Kb in humans) channels on their basolateral membrane, respectively. Both channels are thought to play important roles in controlling systemic water-electrolyte balance and blood pressure. However, little is known about mechanisms regulating activity of Kir4.1/5.1 and ClC-K2/b. Here, we employed patch clamp analysis at the single channel and whole cell levels in freshly isolated mouse collecting ducts to investigate regulation of Kir4.1/5.1 and ClC-K2/b by dietary K+ and Cl- intake. Treatment of mice with high K+ and high Cl- diet (6% K+, 5% Cl-) for 1week significantly increased basolateral K+-selective current, single channel Kir4.1/5.1 activity and induced hyperpolarization of basolateral membrane potential in principal cells when compared to values in mice on a regular diet (0.9% K+, 0.5% Cl-). In contrast, basolateral Cl--selective current and single channel ClC-K2/b activity was markedly decreased in intercalated cells under this condition. Substitution of dietary K+ to Na+ in the presence of high Cl- exerted a similar inhibiting action of ClC-K2/b suggesting that the channel is sensitive to variations in dietary Cl- per se. Cl--sensitive with-no-lysine kinase (WNK) cascade has been recently proposed to orchestrate electrolyte transport in the distal tubule during variations of dietary K+. However, co-expression of WNK1 or its major downstream effector Ste20-related proline-alanine-rich kinase (SPAK) had no effect on ClC-Kb over-expressed in Chinese hamster ovary (CHO) cells. Treatment of mice with high K+ diet without concomitant elevations in dietary Cl- (6% K+, 0.5% Cl-) elicited a comparable increase in basolateral K+-selective current, single channel Kir4.1/5.1 activity in principal cells, but had no significant effect on ClC-K2/b activity in intercalated cells. Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the stimulatory actions of high K+ intake on Kir4.1/5.1 channels in principal cells but was ineffective to alter ClC-K2/b activity and basolateral Cl- conductance in intercalated cells. In summary, we report that variations of dietary K+ and Cl- independently regulate basolateral potassium and chloride conductance in principal and intercalated cells. We propose that such discrete mechanism might contribute to fine-tuning of urinary excretion of electrolytes depending on dietary intake.

KCNJ10 determines the expression of the apical Na-Cl cotransporter (NCC) in the early distal convoluted tubule (DCT1).

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10(+/+) mice (WT). This 40-pS K channel is absent in homozygous Kcnj10(-/-) (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism.

Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1

Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-I(sc)). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-I(sc) by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-I(sc). Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca(2+)](i)) in T84 cells and AMG-9810 blocked the rise in [Ca(2+)](i) induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1.

Immediate and short-term consequences of secondhand smoke exposure on the respiratory system

This review critically evaluates the existing biological evidence regarding the immediate and short-term respiratory consequences of secondhand smoke (SHS). A 1-h exposure to SHS at bar/restaurant levels generates a marked inflammatory reaction and significant decrements on lung function. These deleterious effects of SHS are exacerbated when physical activity follows the SHS exposure, particularly in less fit individuals. The main respiratory effect mechanisms of SHS include a direct induction of growth factors resulting in airway remodelling and alterations in nitric oxide regulation. Pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance may be of therapeutic benefit in patients with diseases related to SHS exposure. Moreover, treatment with statins has shown beneficial effects towards preventing the SHS-induced pulmonary hypertension, vascular remodelling, and endothelial dysfunction. Based on recently discovered evidence, even brief and short-term exposures to SHS generate significant adverse effects on the human respiratory system. Future research directions in this area include the concentrations of tobacco smoke constituents in the alveolar milieu following SHS exposure, individual susceptibility to SHS, as well as pharmacological treatments for reversing the SHS-induced airway remodelling.

A Biophysical Model for Integration of Electrical, Osmotic, and pH Regulation in the Human Bronchial Epithelium

A dynamical biophysical model for the functioning of an epithelium is presented. This model integrates the electrical and osmotic behaviors of the epithelium, taking into account intracellular conditions. The specific tissue modeled is the human bronchial epithelium, which is of particular interest, as it is the location of the most common lethal symptoms of cystic fibrosis. The model is implemented in a modular form to facilitate future application of the code to other epithelial tissue by inputting different transporters, channels, and geometric parameters. The model includes pH regulation as an integral component of overall regulation of epithelial function, through the interdependence of pH, bicarbonate concentration, and current. The procedures for specification, the validation of the model, and parametric studies are presented using available experimental data of cultured human bronchial epithelium. Parametric studies are performed to elucidate a), the contribution of basolateral chloride channels to the short-circuit current functional form, and b), the role that regulation of basolateral potassium conductance plays in epithelial function.

Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells

Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.

Effect of nitric oxide on electrolyte transport across the porcine proximal colon.

The effect of nitric oxide (NO) on ion transport in the porcine proximal colon was investigated in slide-stripped epithelia mounted in Ussing chambers. The serosal addition of the NO-donors sodium nitroprusside (SNP, 0.5 mM) or S-nitroso-N-acetylpenicillamine (SNAP, 0.5 mM) induced a steep increase of short-circuit current ( I(sc)). The stimulatory effect of SNP on I(sc) could not be blocked by piroxicam or tetrodotoxin. Potassium channel inhibitors (quinidine, tetraethylammonium or barium) added serosally reduced the SNP- or SNAP-induced increases of I(sc). In chloride-free solutions, the SNP-induced increase of I(sc) was smaller than in chloride-containing solutions. Cl(- )and Na(+) flux measurements demonstrated that SNP diminished Cl(-) and Na(+) net absorption. Pre-treatment with barium was able to block the inhibitory effect of SNP on NaCl net absorption totally. NO effects on paracellular pathways were assessed by measuring flux rates of [(14)C]-D-mannitol. SNP did not change unidirectional D-mannitol flux rates. In conclusion, NO inhibits NaCl net absorption in the proximal colon of pigs by acting directly on the enterocyte. The antiabsorptive (and/or prosecretory) effect of NO depends on a functional basolateral potassium conductance.

Colocalization of KCNQ1/KCNE channel subunits in the mouse gastrointestinal tract.

The KCNQI potassium channel alpha-subunit can associate with various KCNE beta-subunits that drastically influence channel gating. Here we show that in the mouse gastrointestinal tract KCNQ1 is prominently expressed in stomach, small intestine and colon, while KCNE3 is expressed in the colon and to a lesser extent in small intestine. Immunostaining revealed that KCNQ1 colocalizes with KCNE3 in the basolateral membranes of crypt cells of the colon and small intestine. Together with the previously shown electrophysiological properties of KCNQ1/KCNE3 channels, this strongly suggests that they form the basolateral potassium conductance that is required for transepithelial cAMP-stimulated chloride secretion. In the stomach, KCNQ1 is expressed together with the H+/K+-ATPase in the luminal membrane of acid-secreting parietal cells of gastric glands. KCNE2, but neither KCNE1 nor KCNE3 was detected in the stomach by Northern analysis. Similar to KCNQ1, KCNE2 was present in gastric glands in only a subset of cells that probably represent parietal cells. The coexpression of KCNQ1 and KCNE2 in HEK293 cells yielded potassium currents that were open at resting voltages, suggesting that these heteromeric channels may underlie the apical potassium conductance in acid-secreting parietal cells that is necessary for the recycling of potassium ions during acid secretion via the H+/K+-ATPase.

Basolateral potassium channels and epithelial ion transport.

Basolateral potassium channels and epithelial ion transport.

A Role for Protein Kinase Cε in the Inhibitory Effect of Epidermal Growth Factor on Calcium-stimulated Chloride Secretion in Human Colonic Epithelial Cells

Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T(84) colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKCalpha, gamma, epsilon, eta, mu, lambda/iota, and zeta were expressed in T(84) cells. Ro318220 (an inhibitor active against PKCepsilon, 10 micrometer) but not Gö6983 (an inhibitor active against PKCzeta, 10 micrometer) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKCepsilon from the cytoplasm to the membrane. Wortmannin (50 micrometer) and LY294002 (20 nm), which are PI 3-kinase inhibitors that by themselves had no effect on PKCepsilon activity, significantly suppressed PKCepsilon translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P(2) increased membrane-associated PKCepsilon and reduced carbachol-induced (86)Rb(+) efflux. Antisense oligonucleotides against PKCepsilon decreased PKCepsilon mass and prevented the inhibitory effect of EGF on carbachol-induced (86)Rb(+) efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKCepsilon mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.

Two types of K(+) channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine.

The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, K(ATP), was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 +/- 5 pS (n = 15) and an outward slope conductance of 13 +/- 6 pS (n = 15). The K(ATP) channel open probability (P(o)) is low, 0.15 +/- 0.06 (n = 15) at a -V(p) = -100 mV (V(p) is the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (tau(o) = 5.0 +/- 1.9 ms, n = 6) and two closed time constants (tau(C1) = 5. 2 +/- 1.5 ms, tau(C2) = 140 +/- 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 +/- 2 pS (n = 4). Its P(o) is voltage independent and averages 0.67 +/- 0.03 (n = 4) at -V(p) = -80 mV. Both its open time and closed time distributions are described by a single time constant (tau(o) = 96 +/- 19 ms, tau(C) = 10.5 +/- 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas K(ATP) channel P(o) decreases by 75% (from 0.17 +/- 0.06 to 0.04 +/- 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability K(ATP) channel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

Neuropeptide Y inhibits ion secretion in intestinal epithelium by reducing chloride and potassium conductance.

Neuropeptide Y (NPY) is probably the most abundant neuropeptide, with a plethora of central as well as peripheral effects, including its proabsorptive action in the gastro-intestinal tract. The effects of NPY on electrical parameters related to three different pathways stimulating ion secretion were investigated using the human intestinal cell line HT29cl.19A. Transepithelial potential and resistance were measured with the preparation maintained in a horizontal Ussing chamber, allowing simultaneous measurement of the membrane potential and determination of the fractional resistance of the apical cell membrane. It was found that application of NPY, after the adenylyl-cyclase-activating drug forskolin, resulted in complete inhibition of forskolin-induced effects within approximately 20 min. The secretion stimulated by adenosine appeared to be insensitive to NPY. The acetylcholine analogue carbachol stimulates ion secretion by increasing intracellular free calcium concentrations ([Ca2+]i) which activates the basolateral potassium (K+) conductance. NPY caused 50% inhibition of the effect of carbachol. Measurements of [Ca2+]i showed that NPY inhibited the carbachol-induced rise in [Ca2+]i, which correlates with the reduced activation of basolateral K+ channels. From this study we conclude that NPY inhibits cAMP-stimulated as well as Ca2+-stimulated secretion via a reduction in the apical Cl- and basolateral K+ conductance. This double effect makes NPY an effective proabsorptive peptide.

Inhibition of basolateral potassium conductance by taurine in the proximal convoluted tubule.

The effect of taurine on the electrophysiological properties of the basolateral membrane of the rabbit proximal convoluted tubule was examined. Short-duration isosmotic pulses of 40 mM taurine in the bath solution induced basolateral membrane depolarizations (delta Vbl) of 6.44 +/- 0.5 mV, which were reduced by 58% in absence of Na+. In presence of barium and quinine, delta Vbl values were reduced by 55% in Na(+)-containing bath solutions and were completely abolished in Na(+)-free solutions. Continuous addition of taurine into the bath solutions for a period of 5 min induced 1) a decrease in the partial conductance of the basolateral membrane to K+ (tK) from 0.39 to 0.23; 2) an increase in the partial conductance to Cl- (tcl) from 0.055 to 0.172; 3) a rapid and transient increase of the partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.37 to 0.52, followed by a progressive decrease to 0.29; and 4) a depolarization of the basolateral membrane of 16 mV. The absolute membrane conductance mediated by the Na-HCO3 cotransporter was not initially affected by taurine, whereas that to K (GK) initially decreased by a maximal factor of 2 after 1 min, followed by a partial recovery after 5 min, and that to Cl (GCl) increased by a factor of 4. Addition of taurine after a hypotonicity-induced cell swelling produced an inhibition of GK comparable to the one observed under isotonic conditions. These results demonstrate the presence of an electrogenic Na-dependent transport of taurine and indicate that taurine inhibits GK.

Effects of glucocorticoid and mineralocorticoid on potassium transport in the rat medullary thick ascending limb of Henle's loop

Potassium transport in the thick ascending limb is conducted by heterogeneous cells to opposite directions. Effects of glucocorticoids and mineralocorticoids on potassium transport of the rat medullary thick ascending limb (MTAL) were examined by in vitro microperfusion technique by measuring net potassium flux (JK), and apparent potassium conductances in the apical and basolateral membranes of two cell populations. Segments of MTAL were obtained from four groups of rats: sham operated control rats, adrenalectomized rats (ADX), adrenalectomized rats treated with dexamethasone (DEX), and adrenalectomized rats treated with aldosterone (ALDO). Fractional urinary potassium excretion was reduced by ADX and partially recovered by either DEX or ALDO. JK of the isolated perfused MTAL was markedly decreased by ADX from 5.13 to 1.94 pmol/min/mm. It was partially recovered by DEX (3.36 pmol/min/mm), but not by ALDO (2.07 pmol/min/mm). Random impalement of MTAL cells with a microelectrode in the control group revealed two cell populations; 76% was high basolateral conductance cell (HBC) and 24% was low basolateral conductance cell (LBC). In the ADX group, the basolateral potassium conductance of the HBC cell was markedly reduced, whereas the apical membrane potassium conductance of the HBC cell was increased. These changes were recovered in the DEX group, but not in the ALDO group. Potassium conductances of the apical and basolateral membranes in the LBC were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Basolateral K channel activated by carbachol in the epithelial cell line T84.

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.