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.
Mineralocorticoid receptor (MR)-deficient mice were generated by gene targeting. These animals had a normal prenatal development. During the first week of life, MR-deficient (؊͞؊) mice developed symptoms of pseudohypoaldosteronism. They finally lost weight and eventually died at around day 10 after birth from dehydration by renal sodium and water loss. At day 8, ؊͞؊ mice showed hyperkalemia, hyponatremia, and a strong increase in renin, angiotensin II, and aldosterone plasma concentrations. Methods were established to measure renal clearance and colonic transepithelial Na ؉ reabsorption in 8-day-old mice in vivo. The fractional renal Na ؉ excretion was elevated >8-fold. The glomerular filtration rate in ؊͞؊ mice was not different from controls. The effect of amiloride on renal Na ؉ excretion and colonic transepithelial voltage reflects the function of amiloide-sensitive epithelial Na ؉ channels (ENaC). In ؊͞؊ mice, it was reduced to 24% in the kidney and to 16% in the colon. There was, however, still significant residual ENaC-mediated Na ؉ reabsorption in both epithelia. RNase protection analysis of the subunits of ENaC and (Na ؉ ؉ K ؉ )-ATPase did not reveal a decrease in ؊͞؊ mice. The present data indicate that MR-deficient neonates die because they are not able to compensate renal Na ؉ loss. Regulation of Na ؉ reabsorption via MR is not achieved by transcriptional control of ENaC and (Na ؉ ؉ K ؉ )-ATPase in RNA abundance but by transcriptional control of other as yet unidentified genes. MR knockout mice will be a suitable tool for the search of these genes.
Mammalian cell membranes harbor several types of chloride channels, chloride-cation symporters/cotransporters, and several classes of anion exchangers/antiporters. These transport systems subserve different cellular or organismic functions, depending on the nature of the cell, the spatial organization of transporters, and their functional interplay. Chemical probing has played a central role in the structural and functional delineation of the various anion transport systems. The design of specific probes or their selection from existing sources coupled with their judicious application to the most appropriate biological system had led to the identification of specific anion transporters and to the elucidation of the underlying molecular transport mechanism. In many instances, chemical probing has remained the major or exclusive analytical tool for the functional definition or identification of a given transport system, particularly for discerning among the various anion transporters which operate in highly heterogeneous cell membrane systems. This work critically reviews the present state of the chemical armamentarium available for the most common anion transporters found in mammalian cell membranes. It encompasses the description of the most useful or commonly used probes in terms of their chemical, biochemical, physiological, and pharmacological properties. The review deals primarily with what chemical probes tell about anion transporters and, most importantly, with the limitations inherent in the use of probes in transport studies.
On the basis of our findings with diphenylamine-2-carboxylate we have searched for compounds which possess an even higher affinity for the Cl(-)-channels in the basolateral membrane of the thick ascending limb of the loop of Henle. To quantity the inhibitory potency, we performed measurements of the equivalent short circuit current, corresponding to the secondary active transport of Cl- and measurements of the voltage across the basolateral membrane. A survey of 219 compounds reveals that relatively simple modifications in the structure of diphenylamine-2-carboxylate led to very potent blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate which inhibits the short circuit current half maximally (IC50) at 8 X 10(-8) mol/l. A comparison of the structural formula and the respective IC50 values leads to several empirical conclusions: The potent compounds are lipophilic due to the apolar residue (e.g. phenyl- or cycloalkyl group). Replacing this part of the molecule by an aliphatic chain (up to 4 C-atoms) leads to inactive compounds. Most of the inhibitors are secondary amines. Linking other than with -NH- between the phenyl ring and the benzoic acid results in inactive compounds. Tertiary amines, such as in case of 2-(N,N-diphenylamine)benzoic acid or N-methylphenylamine-benzoic acid are poorly active. The carboxylate group of the benzoate moiety must be in ortho position to the amino group. Introduction of substituents into the benzoate moiety e.g. -NO2 (in meta position to the carboxylate group), or by -Cl (in para position to the carboxylate group) results in an increase of inhibitory potency. A -CH2-, -C2H4-, -C3H6- spacer between the amino bridge and the phenyl ring increases the affinity for the Cl(-)-channel by several orders of magnitude. The above described structure activity relationship renders it likely that these chloride channel blockers possess several sites of interaction: The negatively charged carboxylate group, the secondary amine group which probably carries a positive partial charge, and for the very potent agents (nos. 130, 143, 144, and 145) an additional negative partial charge at the respective -Cl or -NO2 substituent. Finally, also an apolar interaction with an cycloalkyl or cycloaryl residue seems to be required, and this site of interaction has a defined spacing from the secondary amino nitrogen.
Cl−secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca2+concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current ( I sc) [change (Δ) in I sc = −79.4 ± 7.5 μA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl− exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (Δ I sc = −42.4 ± 7.2 μA/cm2) that was inhibited by basolateral trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Δ I sc = 167.7 ± 32.7 μA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive I sc was probably due to activation of apical K+channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I sc. Under these conditions, CCH induced a large further increase in lumen-negative I sc(Δ I sc = −100.0 ± 21.0 μA/cm2). We conclude that CCH acting via [Ca2+]ican induce Cl− secretion only in the presence of cAMP, i.e., when luminal Cl− channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.
Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl Ϫ conductance, are characterized by an enhanced Na ϩ conductance. In this study we have examined the Na ϩ conductance in human respiratory tract by measuring transepithelial voltage and resistance (V te , R te ) and by assessing membrane voltages (V m ) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 mol/liter) equivalent short circuit current (I sc ϭ V te /R te ) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 mol/liter) a significant depolarization of V m corresponding to the cAMP-dependent activation of a Cl Ϫ conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N -methyl-D -glucamine on V m were attenuated in the presence of forskolin. Also the amiloride-inhibitable I sc was significantly reduced by forskolin (1 mol/liter) and isobutylmethylxanthine (IBMX; 100 mol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na ϩ channels in human airways. This downregulation of epithelial Na ϩ channels is absent in CF airways, leading to hyperabsorption and to the characteristic increase in mucus viscosity. ( J. Clin. Invest. 1998. 102: 15-21.)
In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n = 260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60 +/- 6 pS (n = 24) and 31 +/- 7 pS (n = 4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72 +/- 4 pS (n = 37) at a clamp voltage of 0 mV. The permeability was 0.33 +/- 0.02 . 10(-12) cm3/s. The selectivity sequence of this channel was: K+ = Rb+ = NH4+ = Cs+ greater than Li+ much greater than Na+ = 0; the conductance sequence was K+ much greater than Li+ much greater than Rb+ = Cs+ = NH4+ = Na+ = 0. In excised patches Rb+, Cs+ and NH4+ when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5 +/- 1.5 ms, n = 18) and by two closed (1.4 +/- 0.1 and 14 +/- 2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mumol/l -0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10-100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities greater than 10(-7) mol/l and by adenosine triphosphate greater than or equal to 10(-4) mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by less than or equal to 0.2 units).(ABSTRACT TRUNCATED AT 400 WORDS)
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