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M Bleich

Publications and source records attributed to M Bleich.

At least 19 recordsLinked to original sources
PubMed2005

Renal accumulation and clearance of advanced glycation end-products in type 2 diabetic nephropathy: effect of angiotensin-converting enzyme and vasopeptidase inhibition.

AIMS/HYPOTHESIS: Renal accumulation of AGEs may contribute to the progression of diabetic nephropathy. We evaluated the effect of ramipril (a pure ACE inhibitor) and AVE7688 (a dual inhibitor of ACE and neutral endopeptidase) on renal accumulation of the advanced glycation end-product (AGE) 3-deoxyglucosone-imidazolone, carboxymethyllysine (CML) and pentosidine, and on clearance of CML in type 2 diabetes. METHODS: Male Zucker diabetic fatty rats (ZDF, Gmi-fa/fa) rats were treated from age 10 to 37 weeks with ramipril (1 mg.kg(-1).day(-1)), AVE7688 (45 mg.kg(-1).day(-1)) or without drug. Ramipril and AVE7688 reduced albuminuria by 30 and 90%, respectively. RESULTS: ZDF rats showed increased renal accumulation of the AGE subtypes 3-deoxyglucosone-imidazolone, pentosidine and CML by about 40, 55 and 55%, respectively compared with heterozygous, non-diabetic control animals at the age of 37 weeks. AVE7688 but not ramipril attenuated the renal accumulation of 3-deoxyglucosone-imidazolone, pentosidine and CML and improved CML clearance in ZDF rats. During glycation reactions in vitro, AVE7688 also demonstrated potent chelating activity and inhibited metal-catalysed formation of pentosidine and CML. CONCLUSIONS/INTERPRETATION: Improved AGE clearance and direct inhibition of AGE formation by chelation may contribute to reduced accumulation of renal AGEs and to the nephroprotective effects of vasopeptidase inhibition in type 2 diabetes.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels.

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC(50) values of 5.4+/-0.7 microM and 6.2+/-0.4 microM respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC(50) of 1.1+/-0.2 microM. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation ( tau(inact)), and the integral current was inhibited with an IC(50) of 3.4+/-0.5 microM. At 10 microM AVE0118 tau(inact) decreased from 9.3+/-0.6 ms ( n=8, control) to 3.0+/-0.3 ms ( n=8). The K(ACh) current was investigated in isolated pig atrial myocytes by application of 10 microM carbachol. At a clamp potential of -100 mV the I(KACh) was half-maximally blocked by 4.5+/-1.6 microM AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at -100 mV. Effects on the I(Kr) current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 microM. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I(Kr) tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I(Ks), I(KATP) (recorded in guinea pig ventricular myocytes), and L-type Ca(2+) (recorded in pig atrial myocytes) were blocked by 10 microM AVE0118 by 10+/-3% ( n=6), 28+/-7% ( n=4), and 22+/-13% ( n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K(+) channels I(Kur), I(to) and I(KACH). This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.

Animals↗
PubMed2004

Inhibitors of ATP-sensitive potassium channels in guinea pig isolated ischemic hearts.

During heart ischemia, ATP-sensitive potassium channels in the sarcolemmal membrane (sarcK(ATP)) open and cause shortening of the action potential duration. This creates heterogeneity of repolarization, being responsible for the development of re-entry arrhythmias and sudden cardiac death. Therefore, the aim is to develop selective blockers of the cardiac sarcK(ATP) channel. In the present study we established an in vitro model and classified 5 K(ATP) channel inhibitors with respect to their potency and selectivity between cardiomyocytes and the coronary vasculature and compared the results with inhibition of Kir6.2/SUR2A channels expressed in HEK293 cells, recorded with the Rb(+)-efflux methods. We used Langendorff-perfused guinea pig hearts, where low-flow ischemia plus hypoxia was performed by reducing the coronary flow (CF) to 1.2 ml/min and by gassing the perfusion solution with N(2) instead of O(2). Throughout the experiment, the monophasic action potential duration at 90% repolarization (MAPD(90)) was recorded. In separate experiments, high-flow hypoxia was produced by oxygen reduction in the perfusate from 95% to 20%, which caused an increase in the coronary flow. Under normoxic conditions, the substances glibenclamide, repaglinide, meglitinide, HMR 1402 and HMR 1098 (1 microM each) reduced the CF by 34%, 38%, 19%, 12% and 5%, respectively. The hypoxia-induced increase in CF was inhibited by the compounds half-maximally at 25 nM, approximately 200 nM, 600 nM, approximately 9 microM and >100 microM, respectively. In control experiments after 5 min low-flow ischemia plus hypoxia, the MAPD(90) shortened from 121+/-2 to 99+/-2 ms ( n=29). This shortening was half-maximally inhibited by the substances at concentrations of 95 nM, 74 nM, 400 nM, 110 nM and 550 nM, respectively. In HEK293 cells the Rb(+)-efflux through KIR6.2/SUR2A channels was inhibited by the compounds with IC(50) values of 21 nM, 67 nM, 205 nM, 60 nM and 181 nM, respectively. In summary, the present data demonstrate that the sulfonylurea glibenclamide, and the carbamoylbenzoic acid derivatives repaglinide and meglitinide are unselective blockers of K(ATP) channels in cardiac cells and in the cardiac vascular system, whereas the sulfonylthioureas HMR 1402, and especially HMR 1098 selectively blocked the cardiac sarcK(ATP) channel. Blockade of Kir6.2/SUR2A channels in HEK293 cells occurred with comparable efficacy as in the cardiac tissue, indicating that the expression system is suited for screening for novel inhibitors.

ATP-Binding Cassette Transporters↗
PubMed2003

The vasopeptidase inhibitor AVE7688 ameliorates Type 2 diabetic nephropathy.

AIM/HYPOTHESIS: Pharmacological inhibition of the renin angiotensin system has proven clinical efficacy in nephropathies of various origins, including diabetic nephropathy. We tested the effects of the dual inhibition of both angiotensin converting enzyme and neutral endopeptidase by the vasopeptidase inhibitor AVE7688 in an animal model of Type 2 diabetic nephropathy. METHODS: We treated 56 obese Zucker diabetic fatty (ZDF, Gmi-fa/fa) rats aged 34-weeks with either placebo ( n=9) or the vasopeptidase inhibitor AVE7688 in four different doses (each n=9; 3, 10, 30, or 60 mg/kg/d in chow). We used 11 heterozygous (+/fa) rats which received placebo and served as non-diabetic, lean controls. Urinary albumin/creatinine ratio was assessed as a marker of nephropathy at baseline (age 34-weeks) and after 10 weeks of chronic treatment. RESULTS: All obese animals had established diabetes mellitus that was not influenced by AVE7688 (HbA(1c) >12%, stable in all dose groups). There was massive albuminuria in the homozygous ZDF rats (albumine/creatinine ratio >20 mg/mg vs minimal albuminuria in lean controls) that was decreased by AVE7688 in a dose dependent manner (Placebo 2.0+/-4.4 vs 11.9+/-1.8, 13.4+/-0.7, 13.6+/-2.8, and 19.8+/-2.8 mg/mg in the 3, 10, 30, and 60 mg/kg/d groups, respectively; all treatment groups p<0.05 vs Placebo). CONCLUSION/INTERPRETATION: AVE7688 ameliorates proteinuria in Zucker diabetic fatty rats with established diabetes mellitus. Vasopeptidase inhibition represents an effective novel therapeutic principle for intervention in Type 2 diabetic nephropathy independent of metabolic control.

Albuminuria↗
PubMed2002

The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.

Animals↗
PubMed2001

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↗
PubMed2001

Synthesis and activity of novel and selective I(Ks)-channel blockers.

Since the discovery of the I(Ks)-potassium channel as the slowly activating component of the delayed rectifier current (I(k)) in cardiac tissue, the search for blockers of this current has been intense. During the screening of K(ATP)-channel openers of the chromanol type we found that chromanol 293B was able to block I(Ks). Chromanol 293B is a sulfonamide analogue of the K(ATP)-channel openers but had no activity on this target. Experiments were initiated to improve the activity and properties based on this lead compound. As a screening model we used Xenopus oocytes injected with human minK (KCNE1). Variations of the aromatic substituent and the sulfonamide group were prepared, and their activity was evaluated. We found that the greatest influence on activity was found in the aromatic substituents. The most active compounds were alkoxy substituted. We chose HMR1556 ((3R, 4S)-(+)-N-[-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methyl-ethanesulfonamide) 10a for development as an antiarrhythmic drug. The absolute configuration, resulting from an X-ray single-crystal structure analysis, was determined.

Animals↗
PubMed2001

Regulation of slowly activating potassium current (I(Ks)) by secretin in rat pancreatic acinar cells.

1. The secretagogue-activated K(+) conductance is indispensable for the electrogenic Cl(-) secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP-mediated secretagogues on the slowly activating voltage-dependent K(+) current (I(Ks)) of rat pancreatic acinar cells (RPAs) with the whole-cell patch clamp technique. 2. Upon depolarization, RPAs showed I(Ks) superimposed upon the instantaneous background outward current. Secretin (5 nM), vasoactive intestinal peptide (5 nM), forskolin (5 microM), isoprenaline (10 microM) or 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM) increased the amplitude of I(Ks) two- to fourfold. 3. The physiological concentration of secretin (50 pM) had a relatively weak effect on I(Ks) (160 % increase), which was significantly enhanced by transient co-stimulation with carbachol (CCh) (10 microM). However, the secretin-induced production of cAMP, which was measured by enzyme-linked immunosorbent assay, was not augmented by co-stimulation with CCh. 4. This study is the first to demonstrate the regulation of K(+) channels in RPAs by cAMP-mediated agonists. The I(Ks) channel is a common target for both Ca(2+) and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates I(Ks), which provides an additional driving force for Cl(-) secretion.

1-Methyl-3-isobutylxanthine↗
PubMed2001

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↗
PubMed2001

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↗
PubMed2001

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↗
PubMed2001

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↗
PubMed2001

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

Animals↗
PubMed2001

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↗
PubMed2001

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↗
PubMed2001

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↗
PubMed2000

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↗