Function of K+ channels in the intestinal epithelium.
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Animals↗
Biomedical subjects
R Warth
Publications and source records attributed to R Warth.
The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse.
KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.
Animals↗
The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea.
The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.
Adenosine Triphosphate↗
Cystic fibrosis and CFTR.
Cystic fibrosis (CF) is a complex disease affecting epithelial ion transport. There are not many diseases like CF that have triggered such intense research activities. The complexity of the disease is due to mutations in the CFTR protein, now known to be a Cl(-) channel and a regulator of other transport proteins. The various interactions and the large number of disease-causing CFTR mutations is the reason for a variable genotype-phenotype correlation and sometimes unpredictable clinical manifestation. Nevertheless, the research of the past 10 years has resulted in a tremendous increase in knowledge, not only in regard to CFTR but also in regard to molecular interactions and completely new means of ion channel and gene therapy.
Cystic Fibrosis↗
Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome.
The voltage-dependent K(+) channel responsible for the slowly activating delayed K(+) current I(Ks) is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K(+) and Na(+) intakes. On a normal K(+) diet, homozygous kcne1(-/-) mice exhibit signs of chronic volume depletion associated with fecal Na(+) and K(+) wasting and have lower plasma K(+) concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K(+) diets or stimulated by low Na(+) diets, a high K(+) diet provokes a tremendous increase of plasma aldosterone levels in kcne1(-/-) mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K(+) in kcne1(-/-) mice. This exacerbated aldosterone production in kcne1(-/-) mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I(Ks) may directly participate in the control of aldosterone production by plasma K(+). These results, which show that KCNE1 and I(Ks) are involved in K(+) homeostasis, might have important implications for patients with I(Ks)-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.
Aldosterone↗
Cloning and function of the rat colonic epithelial K+ channel KVLQT1.
KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.
1-Methyl-3-isobutylxanthine↗
Induction of the epithelial Na+ channel via glucocorticoids in mineralocorticoid receptor knockout mice.
Epithelial Na+ channel (ENaC) activity in kidney and colon is stimulated by aldosterone acting on the mineralocorticoid receptor (MR). MR and the glucocorticoid receptor (GR) show high homology in their DNA-binding domain and have similar affinities to mineralo- and glucocorticoids. We therefore asked whether the glucocorticoid-mediated activation of ENaC is restricted to the presence of MR and used the MR knockout mouse model to address this question. Due to their MR deficiency and the consecutive reduction of ENaC activity these mice die as neonates, and even after appropriate substitution therapy adult MR knockout mice suffer from high Na+ loss and hyperkalemia. In the present study, glucocorticoid treatment restored plasma K+ and almost normalized the fractional excretions of Na+ (FENa+) and K+ (FEK+) in adult salt-substituted MR knockout mice, while the effect of amiloride on FENa+ and FEK+ was augmented in these animals. In order to estimate ENaC activity, measurements of transepithelial equivalent short-circuit current (Isc) were performed. Glucocorticoids induced an amiloride-sensitive Na+ absorption in renal cortical collecting duct and distal colon of MR-/- of about 25% and 50% of the currents observed in glucocorticoid-treated wild-type mice, respectively. In the colon glucocorticoid treatment increased the mRNA abundance of all three ENaC subunits, in the kidney only alpha-ENaC was increased. The regulation of ENaC expression was the same in both genotypes and thus irrespective of the presence of MR. These data show that MR is no prerequisite for the activation of ENaC transcription and activity, and that the respective mechanisms can be stimulated via GR.
Amiloride↗
The cardiac K+ channel KCNQ1 is essential for gastric acid secretion.
BACKGROUND & AIMS: Gastric H+ secretion via the H+/K+-adenosine triphosphatase is coupled to the uptake of K+. However, the molecular identity of luminal K+ channels enabling K+ recycling in parietal cells is unknown. This study was aimed to investigate these luminal K+ channels. METHODS: Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. RESULTS: We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a K+ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with H+/K+-adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. CONCLUSIONS: We identified KCNQ1 as the missing luminal K+ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.
Amino Acid Sequence↗
Characterisation of the rat SK4/IK1 K(+) channel.
BACKGROUND AND AIMS: The Ca(2+)-activated K(+) channel rSK4 is the rat homologue of the human SK4/IK1 (KCNN4) channel. In colonic mucosa rSK4 plays a key role during acetylcholin-induced secretion. This study was aimed to characterize the properties of the rat SK4 channel. METHODS: Electrophysiological measurements were performed on rSK4 expressing Xenopus laevis oocytes and rat colonic crypts. Intracellular Ca(2+) activity was assessed by Oregon Green fluorescence measurements. RESULTS: The 10 pS rSK4 expressed in oocytes was Ca(2+)-sensitive and inhibited by calmodulin antagonists. 1-ethyl-2-benzimidazolinone (1-EBIO), a known activator of SK4/IK1 channels, also activated rSK4. 1-EBIO affected the current neither at saturating Ca(2+) activities nor under Ca(2+)-free conditions, but increased the Ca(2+) sensitivity of rSK4. rSK4 was strongly activated by cytosolic ATP. However, PKA itself, PKA inhibitors and mutation of the PKA phosphorylation site (S332A) did not affect channel activity. The PKC activator 1,2-dioctanoyl-sn-glycerol and the PKC inhibitor bisindolylmaleimide also failed to influence rSK4. CONCLUSION: The Ca(2+)-sensitive rSK4 is activated by 1-EBIO probably via facilitation of the Ca(2+)-calmodulin-rSK4 interaction. The strong ATP-activation of rSK4 is likely to be caused by phosphorylation via a yet unknown kinase and might involve additional subunits.
1-Methyl-3-isobutylxanthine↗
A constitutively open potassium channel formed by KCNQ1 and KCNE3.
Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.
Amino Acid Sequence↗
K+ channels and colonic function.
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Animals↗
The very small-conductance K+ channel KvLQT1 and epithelial function.
KvLQT1 (KCNQ1) is a very small conductance K+ channel distributed widely in epithelial and non-epithelial tissues. Its specific biophysical and pharmacological properties are determined by the regulatory subunits IsK (KCNE1) and MiRP2 (KCNE3). In epithelial cells of the inner ear, pancreas, and airways it interacts with IsK to conduct a voltage-gated and slowly activating K+ current. In the colon it coassembles with KCNE3 to conduct an instantaneous and constitutively active K+ current. In Cl- secretory epithelia, such as the colon and pancreas, this K+ channel provides the driving force for Cl- exit and is located in the basolateral membrane. In the inner ear it enables luminal secretion of K+ into the endolymphatic space. The functional relevance of KvLQT1 to epithelial function is revealed by blocking it pharmacologically or by studying animals with a genetic defect for it, which result in the breakdown of colonic Cl- secretion and endolymph production, respectively. KvLQT1 K+ channels are activated via cAMP or Ca2+ and inhibited by the chromanol 293B. Interaction with as yet unknown regulatory subunits may determine the properties of KvLQT1 in the rectal gland and other epithelial tissues in which KvLQT1 is not inhibited by chromanols.
Animals↗
pH regulation in isolated in vitro perfused rat colonic crypts.
We investigated disorders and regulation of cytosolic pH (pHi) in isolated perfused crypts from rat distal colon using the pH-sensitive dye BCECF. This preparation allows distinct examination of either luminal or basolateral transport. The effects of luminal weak organic acids and bases on pHi were examined. The physiological concentrations of both luminal CO2/HCO3- and acetic acid/acetate acidified pHi significantly, but less than when applied from the basolateral side. Corresponding changes (luminal versus basolateral) in pHi were -0.17+/-0.04 versus -0.39+/-0.04, (n=8) and -0.15+/-0.02 versus -0.41+/-0.04, (n=8), respectively. Basolateral versus luminal application of NH3/NH4+ led to a more marked change in pHi, namely 0.35+/-0.03 versus 0.008+/-0.007 pH units, (n=19). The luminal perfusion of NH3/NH4+ was controlled by applying fura-2 acid to the luminal side and at the same time recording fura-2-specific fluorescence. Hence, the influence of luminal acid/base on colonic pHi homeostasis was limited. To examine pHi regulation, we investigated the recovery from an intracellular acid load using the NH3/NH4+ pulse method. Recovery was completely dependent on basolateral Na+, indicating that luminal acid/base transport does not play a major role in pHi homeostasis. The basolateral transporters involved in pHi recovery are probably the EIPA- and HOE694-inhibitable (IC50=0.2 and 2 micromol/l, respectively) Na+/H+ exchanger NHE1 and the DIDS-inhibitable Na+-dependent HCO3- importer.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts.
The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.
Algorithms↗
Deoxycholic acid (DOC) affects the transport properties of distal colon.
Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1-10 micromol/l) induced a lumen-positive current (51+/-3 microA/cm2, 10 micromol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (-76+/-9 microA/cm2, 100 micromol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc, (-58+/-10 microA/cm2, 100 micromol/l, n=6, EC50= 3 micromol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl- secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pre-treated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100 micromol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (4.9+/-0.8 mV, 100 micromol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24+/-7 mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl- secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca(2+)-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl- secretion indirectly.
Amiloride↗
Regulation of the Na+ 2Cl- K+ co-transporter--mechanisms in the rectal gland of Squalus acanthias with implications for the thick ascending limb of Henle.
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Animals↗
The cellular mechanisms of Cl- secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias.
We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [Isc=transepithelial voltage/transepithelial resistance (Rte)] as well as basolateral membrane voltage (Vbl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a "stimulation cocktail" (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in Isc (-31.6+/-7.7 to -316+/-82.2 microA/cm2, n=15). CNP significantly depolarized the luminal membrane from -87. 4+/-1.0 to -82.3+/-2.6 mV (n=12). Vbl was not changed (n=12) but the fractional conductance for K+ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on Isc were not additive (n=5). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02+/-0.05 to 1.32+/-0.05 (n=8) with a time constant in the 1-2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in Isc in the rectal gland. The concomitant fall in Rte corresponds to increases in the luminal membrane Cl- conductance and in the basolateral membrane K+ conductance. The latter effect is probably due to an increase in [Ca2+]i.
Animals↗
Rescue of the mineralocorticoid receptor knock-out mouse.
The mineralocorticoid receptor knock-out mouse (MR-/-), resembling inborn pseudohypoaldosteronism, dies 8-12 days after birth in circulatory failure with all the signs of terminal volume contraction. The present study aimed to examine the functional defects in the kidney and colon in detail and to attempt to rescue these mice. In neonatal (nn) MR-/- the amiloride-sensitive short-circuit current in the colon was reduced to approximately one-third compared to controls (MR+/+ and MR+/-). In isolated in vitro perfused collecting ducts the amiloride-induced hyperpolarization of the basolateral membrane (Vbl) of nn MR-/- was similar to that of controls, but urinary Na+ excretion was markedly increased to 4.3 micromol/day.g (BW). Based on this measured urinary Na+ loss we tried to rescue nn MR-/- mice by injecting NaCl twice daily (3.85 micromol/g BW), corresponding to 22 microliter of isotonic saline/g BW subcutaneously. This regimen was continued until the animals had reached a body mass of 8.5 g. Thereafter, in addition to normal chow and tap water, NaCl drinking water (333 mmol/l) and pellets soaked in 333 mmol/l NaCl were offered. Unlike the untreated nn MR-/- most of these mice survived. The adult animals were examined between days 27 and 41, some were used for breeding. When compared to age-matched controls the growth of MR-/- was delayed until day 20. Then their growth curve increased in slope and reached that of controls. MR-/- retained their Na+-losing defect. Amiloride's effect on urinary Na+ excretion was not significant in MR-/- mice and the effect on Vbl in isolated cortical collecting ducts was attenuated. The renin-producing cells were hypertrophic and hyperplastic. Plasma renin and aldosterone concentrations were significantly elevated in MR-/- mice. These data indicate that MR-/- can be rescued by timely and matched NaCl substitutions. This enables the animals to develop through a critical phase of life, after which they adapt their oral salt and water intake to match the elevated excretion rate; however, the renal salt-losing defect persists.
Absorption↗