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     

Characterization of the Stretch-activated Chloride Channel in Isolated Human Atrial Myocytes

Macroscopic and unitary currents through stretch-activated Cl⁻ channels were examined in isolated human atrial myocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K⁺ and Ca²⁺ conductances were blocked and the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was reduced, application of positive pressure via the pipette activated membrane currents under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by 60 mV per 10-fold change in the external Cl⁻ concentration, indicating that the current was Cl⁻ selective. The current was inhibited by bath application of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 9-anthracenecarboxylic acid (9-AC). β-Adrenergic stimulation failed to activate a Cl⁻ current. In single channel recordings from outside-out patches, positive pressure in the pipette activated the unitary current with half-maximal activation of 14.7 mm Hg at +40 mV. The current-voltage relationship of single channel activity obtained in inside-out patches was linear in symmetrical Cl⁻ solution with the averaged slope conductance of 8.6 ± 0.7 pS (mean ±sd, n= 10). The reversal potential shift of the channel by changing Cl⁻ concentration was consistent with a Cl⁻ selective channel. The open time distribution was best described by a single exponential function with mean open lifetime of 80.4 ± 9.6 msec (n= 9), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 11.5 ± 2.2 msec (n= 9) and that for the slow component of 170.2 ± 21.8 msec (n= 9). Major changes in the single channel activity in response to pressure were caused by changes in the interburst interval. Single channel activity was inhibited by DIDS and 9-AC in a manner similar to whole-cell configuration. These results suggest that membrane stretch induced by applying pressure via the pipette activated a Cl⁻ current in human atrial myocytes. The current was sensitive to Cl⁻ channel blockers and exhibited membrane voltage-independent bursting opening without sensitive to β-adrenergic stimulation. Received: 21 October 1996/Revised: 17 December 1997     

Characterization of Calcium-activated Chloride Channels in Patches Excised from the Dendritic Knob of Mammalian Olfactory Receptor Neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30–75 nS. The dose response curve for calcium exhibited an EC₅₀ of about 26 μm. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between −80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P_Na/P_Cl of 0.034. The halide permeability sequence was P_Cl > P_F > P_I > P_Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN⁻, acetate⁻, and gluconate⁻, with the permeability sequence P_Cl > P_SCN > P_acetate > P_gluconate. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 \A. Received: 16 April 1997/Revised: 3 October 1997     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997     

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 Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 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     

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     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Tonoplast K⁺ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K⁺ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl⁻ on either the vacuolar side or the cytoplasmic side was partly replaced with F⁻, the reversal potential of the 48 pS channel shifted conform to the Cl⁻ equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl⁻ channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10⁻⁸ m) Ca²⁺, then no Cl⁻ channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca²⁺ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl⁻ ions induced opposite rectification properties. Our results show Ca²⁺-activated Cl⁻ channels in the tonoplast of Chara with holding potential dependent rectification. Received: 30 March 1999/Revised: 10 August 1999     

Apical Na+-Cl− Symport in Rabbit Gallbladder Epithelium: A Thiazide-Sensitive Cotransporter (TSC)

Cl⁻ apically enters the epithelium of rabbit gallbladder by a Na⁺-Cl⁻ symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl⁻ conductance (G _Cl ), the symport inhibition might be merely due to a short circuit of the symport by G _Cl rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl⁻ uptake, electrophysiological determinations of intracellular Cl⁻ and Na⁺ activities (a _i,Cl and a _i,Na ) with selective theta microelectrodes and molecular biology methods were used. The ³⁶Cl⁻ uptake proved to be a measurement of the apical unidirectional Cl⁻ influx (J _mc ) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G _Cl activation or abolition. After HCTZ treatment the decrease in a _i,Cl (measured as the initial rate or in 3 min) was larger than the decrease in a _i,Na . The difference was reduced to one third in a group of epithelia in which the elicited G _Cl was reduced to one third; moreover it was abolished in any case when G _Cl was abolished with 10⁻⁴ m SITS. The SITS-insensitive rate of a _i,Cl decrease was equal to that of the a _i,Na decrease in any case. Thus the a _i,Cl decrease displays a component dependent on G _Cl activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G _Cl activation and Na⁺-Cl⁻ symport inhibition. This Na⁺-Cl⁻ symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium. Received: 29 July 1999/Revised: 23 March 2000     

HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium

We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO₃. Experimental data suggest that NaHCO₃ secretion occurs in three steps: (i) accumulation of HCO⁻ ₃ across the basolateral membrane of the duct cell by Na(HCO₃) _n cotransporters, Na⁺/H⁺ exchangers and proton pumps; (ii) secretion of HCO⁻ ₃ across the luminal membrane on Cl⁻/HCO⁻ ₃ antiporters operating in parallel with Cl⁻ channels; and (iii) diffusion of Na⁺ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO⁻ ₃ secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO⁻ ₃-rich fluid, it can only raise the luminal HCO⁻ ₃ concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO₃ by the model it is necessary to: (i) reduce the conductive Cl⁻ permeability and increase the conductive HCO⁻ ₃ permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl⁻/HCO⁻ ₃ antiporters. Under these conditions most of the HCO⁻ ₃ is secreted via a conductive pathway. Based on our data, we propose that HCO⁻ ₃ secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO⁻ ₃ concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO⁻ ₃ to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000     

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     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

An Effect of Ca2+ on the Intrinsic Cl−-conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent²⁺-precipitation technique using either CaCl₂ or MgCl₂. The dependence of the initial [¹⁴C]-d-glucose (or [³H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na⁺ salt, was found to be dependent upon the precipitating divalent cation. With Mg²⁺ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca²⁺ precipitation. When the anion composition of the media was varied (uptake in Cl⁻ media in comparison to gluconate⁻-containing media) it was found that the Cl⁻-dependent component of the initial uptake was markedly depressed with Ca²⁺-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg²⁺ and Ca²⁺ prepared vesicles respectively). When Ca²⁺ was loaded into Mg²⁺ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl⁻ enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of d-glucose uptake by Ca²⁺ in Mg²⁺ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca²⁺ inhibition of d-glucose uptake and restore the enhancement due to Cl⁻ media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca²⁺-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl⁻ vs. gluconate⁻) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC₃-(5) confirmed that Cl⁻ conductance was reduced in Ca²⁺-prepared vesicles. We conclude that a Cl⁻ conductance coexists with Na⁺ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca²⁺ via stimulation of protein phosphatase (PP2B). Received: 14 December 1994/Revised: 27 November 1995     

Further Characteristics of the Ca2+-inactivated Cl− Channel in Xenopus laevis Oocytes

Removal of extracellular Ca²⁺ activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca²⁺-inactivated ion channels. Recently, we identified one of these channels as a Ca²⁺-inactivated Cl⁻ channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent cations Zn²⁺ (half-maximal blocker concentration, K_1/2= 8 μm), Cu²⁺ (K_1/2= 120 μm) and Gd³⁺ (K_1/2= 20 μm). Furthermore, CaIC is inhibited by the specific Cl⁻ channel blocker NPPB (K_1/2≈ 3 μm). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 μm) but insensitive to its high affinity analogue benzamil (100 μm). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous measurements of membrane current (I _m ), conductance (G _m ) and capacitance (C _m ) we demonstrate that Ca²⁺ removal leads to instant activation of CaIC already present in the plasma membrane. Since C _m remains constant upon Ca²⁺ depletion while I _m and G _m increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when oocytes are utilized as an expression system for foreign proteins whose investigations require Ca²⁺-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca²⁺-inactivated cation channels reported by other groups. Received: 18 June 1999/Revised: 13 August 1999     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997