Study of Sugar Binding to the Sucrose-specific ScrY Channel of Enteric Bacteria Using Current Noise Analysis

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-α (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40–50% reduction in TER and a 30% decrease in I _sc (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [¹⁴C]-d-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial ²²Na⁺ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na⁺ and Cl⁻. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [³H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mm apical amiloride reduced the basolateral to apical flux of ²²Na⁺, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na⁺ and Cl⁻. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity. Received: 4 March 1997/Revised: 3 November 1997     

Evidence that Basolateral But Not Apical Membrane Localization of Outwardly Rectifying Depolarization-Induced Cl− Channel in Airway Epithelia

The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl⁻ (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl⁻ current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br⁻≒ I⁻ > Cl⁻. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I _sc ) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl⁻ current is present on the basolateral membrane of airway epithelia. Received: 7 October 1999/Revised: 24 April 2000     

Fish Gill Respiratory Cells in Culture: A New Model for Cl−-secreting Epithelia

Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (I _sc : 1.4 ± 0.3 μA/cm²), a transepithelial voltage (V _t ) of 12.7 ± 2.7 mV (serosal positive) and a high transepithelial resistance (R _t : 12302 ± 2477 Ω× cm²). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased I _sc (11.9 ± 2.5 μA/cm²) and V _t (25.9 ± 1.7 mV) and reduced R _t (4271 ± 568 Ω× cm²) as compared with FBS-treated cells. Apical amiloride addition (up to 100 μm) had no effect on I _sc . The I _sc , correlated with an active Cl⁻ secretion measured as the difference between ³⁶Cl⁻ unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na⁺/K⁺/2Cl⁻ cotransport and a Cl⁻/HCO₃ ⁻ exchanger both responsible for Cl⁻ entry through the basolateral membrane and by apical cAMP-sensitive Cl⁻ channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills. Received: 12 July 1996/Revised: 5 November 1996     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

Properties of two multisubstate Cl− channels from human syncytiotrophoblast reconstituted on planar lipid bilayers

We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation, and Mg²⁺-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated in most cases, a result consistent with the higher permeability for cations as compared with Cl⁻ and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode techniques. In this paper, we report that Cl⁻-selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role of the reported Cl⁻ channels in transplacental ion transport and/or syncytium homeostasis remains to be determined. Received: 17 September/Revised: 12 December 1996     

Mitochondria-rich Cells and Voltage-activated Chloride Current in Toad Skin Epithelium: Analysis with the Scanning Vibrating Electrode Technique

The pathway for the voltage-activated chloride current across isolated toad skin was analyzed using a scanning 2D-vibrating voltage probe technique, which permits discrimination of local current peaks if their origins are more than 50 μm apart. The epithelium was separated from the corial connective tissue after enzymatic digestion with collagenase. Cl⁻ current was activated by voltage clamping the transepithelial potential to 60–100 mV, serosa positive. Activated inward current was between 85 and 450 μA/cm². In more than 25 tissue areas of 150 × 100 μm from 10 animals, which were automatically scanned with the vibrating probe, between 0 and 4 peaks of elevated local current (up to 800 μA/cm²) could be identified in individual fields. The density of current peaks, which were generally located at sites of mitochondria-rich (MR) cells, was less than 10% of the density of microscopically identified MR cells. The total current across individual sites of elevated conductance was 3.9 ± 0.6 nA. Considering the density of peaks, they account for 17 ± 2.5% of the applied transepithelial clamping current. The time course of current activation over previously identified conductive sites was in most cases unrelated to that of the total transepithelial current. Moreover, initially active sites could spontaneously inactivate. The results indicate that detection of elevated current above some MR cells is not sufficient to verify these cells as the pathway for transepithelial voltage-activated Cl⁻ current. Since the major fraction of activated current is apparently not associated with a route through MR cells, channel-like structures in the tight junctions of the paracellular pathway must be considered as an alternative possibility. Current peaks over MR cells could be due to high density of such sites in tight junctions between MR and surrounding principal cells. Improvement of the spatial resolution of the vibrating probe is required to verify this view. Received: 29 May 1997/Revised: 29 September 1997     

A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO⁻ ₃-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO₂/HCO⁻ ₃-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO₂/HCO⁻ ₃-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K⁺ concentration, and the effect of K⁺ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na⁺-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl⁻-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl⁻ conductance plays a key role in controlling both the HCO⁻ ₃ secretory rate and intracellular pH during HCO⁻ ₃ secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO⁻ ₃-transporting mechanisms in pancreatic duct cells. Received: 18 October 1995/Revised: 5 July 1996     

Regulation of Chloride Channels in Secretory Epithelia

Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl⁻ channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular increases in two second messenger systems, cAMP and [Ca²⁺] _i , are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types of Cl⁻ channels found in secretory epithelia. Received: 16 September 1997/Revised: 13 November 1997     

Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl− channel

The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl⁻ channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl⁻ currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced activation of the Cl⁻ currents in both cells. The relation between whole-cell Cl⁻ currents and cell size measured simultaneously showed that volume sensitivity of the Cl⁻ channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase in volume sensitivity of the Cl⁻ channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein does not represent the volume-sensitive Cl⁻ channel but that its expression modulates volume sensitivity of the Cl⁻ channel in a manner independent of its ATPase activity or of the protein kinase C activity. Received: 25 September 1996/Revised: 12 December 1996     

Differential Effects of Aldosterone and Vasopressin on Chloride Fluxes in Transimmortalized Mouse Cortical Collecting Duct Cells

The effects of aldosterone and vasopressin on Cl⁻ transport were investigated in a mouse cortical collecting duct (mpkCCD) cell line derived from a transgenic mouse carrying the SV40 large T antigen driven by the proximal regulatory sequences of the L-pyruvate kinase gene. The cells had features of a tight epithelium and expressed the amiloride-sensitive sodium channel and the cystic fibrosis transmembrane conductance regulator (CFTR) genes. dD-arginine vasopressin (dDAVP) caused a rapid, dose-dependent, increase in short-circuit current (I _sc ). Experiments with ion channel blockers and apical ion substitution showed that the current represented amiloride-sensitive Na⁺ and 5-nitro-2-(3-phenylpropylamino)benzoate-sensitive and glibenclamide-sensitive Cl⁻ fluxes. Aldosterone (5 × 10⁻⁷ m for 3 or 24 hr) stimulated I _sc and apical-to-basal ²²Na⁺ flux by 3-fold. ³⁶Cl⁻ flux studies showed that dDAVP and aldosterone stimulated net Cl⁻ reabsorption and that dDAVP potentiated the action of aldosterone on Cl⁻ transport. Whereas aldosterone affected only the apical-to-basal ³⁶Cl⁻ flux, dDAVP mainly increased the apical-to-basal Cl⁻ flux and the basal-to-apical flux of Cl⁻ to a lesser extent. These results suggest that the discrete dDAVP-elicited Cl⁻ secretion involves the CFTR and that dDAVP and aldosterone may affect in different ways the observed increased Cl⁻ reabsorption in this model of mouse cultured cortical collecting duct cells. Received: 8 January 1998/Revised: 25 March 1998     

Apical Heterotrimeric G-proteins Activate CFTR in the Native Sweat Duct

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl⁻ conductance (CFTR G _Cl ) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G _Cl activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G _Cl in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G _Cl by 44 ± 20 mS/cm² (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G _Cl even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF₄ ⁻) in the cytoplasmic bath activated CFTR G _Cl (increased by 51.5 ± 9.4 mS/cm² in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G _Cl in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G _Cl activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G _Cl activity. Received: 9 June 2000/Revised: 5 October 2000     

Further evidence for the regulation of the tight junction ion selectivity by cAMP in goldfish intestinal mucosa

Summary It has been reported that cAMP controls the transepithelial Cl^– conductance in fish intestine (Bakker, R., Groot, J.A., 1984,Am. J. Physiol. 246:G213–G217; Krasny, E.J., Madara, J.L., DiBona, D.L., Frizzell, R.A., 1983,Fed. Proc. 42:1100). In both studies, the cAMP effect was interpreted as an increase in tight junction Cl^– conductance, because cAMP did not change the membrane potential or membrane resistance ratio. However, the activation of a Cl^– conductance in the membranes of a subset of the epithelial cells might be difficult to discern from an increase in tight junction Cl^– conductance. Here we report experiments that were designed to distinguish a tight junction Cl^– conductance from a membrane Cl^– conductance in a subpopulation of the epithelial cells. The effect of hypotonicity on the cAMP-induced increase in transepithelial conductance showed that cAMP-induced conductance is located in series with the lateral intercellular spaces. Transepithelial serosa to mucosa direct current caused an increase in resistance due to so-called transport number effects. Forskolin abolished the transport number effects, indicating that cAMP increases the Cl^– conductance of the tight junctions. Increasing cAMP did not change mannitol fluxes, whereas Cl^– fluxes more than doubled. Changes in dilution potential and transepithelial resistance demonstrated that the cAMP-induced conductance is specific for Cl^– and Br^– as opposed to I^–, NO ₃ ^– , SO ₄ ^2– and gluconate^–. In contranst, cytochalasin D also decreased the transepithelial resistance and dilution potential in Nagluconate Ringer's. This demonstrates that cAMP acts on the tight junctions in a more specific manner than cytochalasin D.     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

The Ca2+-inactivated Cl− Channel at Work: Selectivity,Blocker Kinetics and Transport Visualization

Removal of extracellular divalent cations activated a Cl⁻ channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca²⁺-inactivated Cl⁻ channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl⁻ channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl⁻-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl⁻ from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca²⁺ removal. In the absence of external Cl⁻ and Ca²⁺, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl⁻ in the millimolar range. In the presence of external Ca²⁺, no emission changes could be observed during the same time period. Chelating external Ca²⁺ in absence of Cl⁻ immediately activated Ca²⁺-inactivated Cl⁻ channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I⁻ > Br⁻ > Cl⁻≫ gluconate as it is also typical for maxi Cl⁻ channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca²⁺-inactivated Cl⁻ channels. However, this egg CaIC revealed an altered sensitivity to external Ca²⁺ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl⁻ channels described up to now. Received: 16 May 1996/Revised: 4 September 1996     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

Effect of Dicyclohexylcarbodiimide (DCCD) on Transport Parameters in the Frog Cornea Epithelium

Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na⁺/K⁺ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I _sc ) conditions. Addition of DCCD to a concentration of 10⁻⁴ m to the tear solution gave a marked decrease in I _sc ; a marked depolarization of the intracellular potential, V _o ; and a significant decrease in the apical membrane fractional resistance, fR _o . There were small and variable although significant changes in the transepithelial conductance, g _t . The effects may be explained by a decrease in the basolateral membrane K⁺ conductance, in combination with a partial inhibition of the Na⁺/K⁺-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl⁻ conductance. Received: 12 August 1999/Revised: 16 November 1999     

Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl⁻ than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br⁻ > Cl⁻ > I⁻≈ F⁻. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br⁻ and Cl⁻ have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl⁻. Our results suggest that channel block involves the interaction of HPO²⁻ ₄ with a site located at 70% of the membrane span. Received: 10 January 1997/Revised: 29 May 1997     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996