Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

K-Cl cotransporters (KCC) playfundamental roles in ionic and osmotic homeostasis. To date, fourmammalian KCC genes have been identified. KCC2 is expressed exclusivelyin neurons. Injection of Xenopus oocytes with KCC2cRNA induced a 20-fold increase in Cl-dependent,furosemide-sensitive K⁺ uptake. Oocyte swelling increasedKCC2 activity 2-3 fold. A canonical tyrosine phosphorylationsite is located in the carboxy termini of KCC2 (R1081-Y1087) andKCC4, but not in other KCC isoforms. Pharmacological studies, however,revealed no regulatory role for phosphorylation of KCC2 tyrosineresidues. Replacement of Y1087 with aspartate or arginine dramaticallyreduced K⁺ uptake under isotonic and hypotonic conditions.Normal or near-normal cotransporter activity was observed when Y1087was mutated to phenylalanine, alanine, or isoleucine. A tyrosineresidue equivalent to Y1087 is conserved in all identified KCCs fromnematodes to humans. Mutation of the Y1087 congener in KCC1 toaspartate also dramatically inhibited cotransporter activity. Takentogether, these results suggest that replacement of Y1087 and itscongeners with charged residues disrupts the conformational state ofthe carboxy terminus. We postulate that the carboxy terminus plays anessential role in maintaining the functional conformation of KCCcotransporters and/or is involved in essential regulatory protein-protein interactions.

     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na⁺-K⁺-2Cl^– cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K⁺-Cl^– cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. bumetanide; Na⁺-K⁺-2Cl^– cotransporter; K⁺-Cl^– cotransporter; Xenopus oocytes     

Electrophysiological Measurements on the Root of Atriplex hastata

The ion contents and membrane potentials of the cells of young,hydroponically cultured seedlings of Atriplex hastata L. var.salina, Wallr. have been measured at several different NaClconcentrations. The total tissue concentrations of Na⁺ and Cl^–increase as external NaCl increases, but there is always a markedexcess of internal Na⁺ over Cl^–; this is balanced by endogenousorganic anion formation with a concomitant extrusion of H⁺ tothe bathing solution. Membrane potentials of the root cells remain essentially invariantwith changes in external NaCl at approx. –130 mV; thereis no evidence of a radial gradient of potential across theroot. The potential seems to contain a cyanide-sensitive electrogeniccomponent, also invariant with NaCl concentration, of about–70 mV, and a diffusion component. The electrogenic componentseems likely to be a H⁺ efflux, probably through a H⁺ uniportATPase.     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP I. REPLACEMENT OF THE CELL SAP WITH ARTIFICIAL SOLUTIONS

A method for replacing the cell sap of Nitella with an artificialsolution was introduced. The technique, which is a modificationof KAMIYA and KURODA'S (1, 2), is applicable not only for isotonicbut also for hypertonic or hypotonic solutions. Photometricdeterminations of K⁺, Na⁺, Ca⁺⁺ and Cl^– proved that thereplacement of the cell sap with the present method is satisfactory.The internodal cell of Nitella, whose cell sap was replacedwith an isotonic solution with a simple composition such asa mixture of KCl, NaCl and CaCl₂, can be kept living at leastfor several days, sometimes even for more than one month. (Received September 6, 1963; )     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d 33.4 g/dl) has only about 5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d 24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d_50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d_50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r² = 0.995) and sickle erythrocytes (r² = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity. phosphorylation; protein phosphatase; urea; cell size; density     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Inhibition of Cl- Channel Activation in Chara corallina Membrane by Lanthanum Ion

We examined a role of Ca²⁺ in the activation of the two majorion channels, i.e., Cl^– and K⁺ channels at the excitationof the characean plasmalemma. The current-voltage relation (I-Vcurve) of the Chara membrane was compared under the ramp voltageclamp condition before and after external application of 20µMof La³⁺ (a Ca²⁺ channel blocker). The transient inward currentcomponent, which is carried mainly by the efflux of Cl^–,disappeared almost completely in about 30 min with La³⁺ treatment.On the other hand, no effect was observed on the late largeoutward current, which is mainly carried by the efflux of K⁺in a large depolarization region (less negative than –50mV). These results suggest that the Cl^– channel in theChara plasmalemma is activated by Ca²⁺ influx, while the K⁺channel is simply activated by depolarization. (Received April 7, 1986; Accepted June 6, 1986)     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

Characterization of a novel voltage-dependent outwardly rectifying anion current in Caenorhabditis elegans oocytes

An inwardly rectifying swelling- and meiotic cell cycle-regulated anion current carried by the ClC channel splice variant CLH-3b dominates the whole cell conductance of the Caenorhabditis elegans oocyte. Oocytes also express a novel outwardly rectifying anion current termed I_Cl,OR. We recently identified a worm strain carrying a null allele of the clh-3 gene and utilized oocytes from these animals to characterize I_Cl,OR biophysical properties. The I_Cl,OR channel is strongly voltage dependent. Outward rectification is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV. Apparent channel open probability is zero at voltages less than +20 mV. The channel has a 4:1 selectivity for Cl^– over Na⁺ and an anion selectivity sequence of SCN^– > I^– > Br^– > Cl^– > F^–. I_Cl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 9-anthracenecarboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid. However, the current is rapidly inhibited by niflumic acid, metal cations including Gd³⁺, Cd²⁺, and Zn²⁺, and bath acidification. The combined biophysical properties of I_Cl,OR are distinct from those of other anion currents that have been described. During oocyte meiotic maturation, I_Cl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte Cl^– homeostasis, development, cell cycle control, and/or ovulation. chloride channel; ovulation; cell cycle; meiotic maturation     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

Cl- secretory effects of EBIO in the rabbit conjunctival epithelium

Experiments were conducted to determine whether the Cl^– secretagogue, 1-ethyl-2-benzimidazolinone (EBIO), stimulates Cl^– transport in the rabbit conjunctival epithelium. For this study, epithelia were isolated in an Ussing-type chamber under short-circuit conditions. The effects of EBIO on the short-circuit current (I_sc) and transepithelial resistance (R_t) were measured under physiological conditions, as well as in experiments with altered electrolyte concentrations. Addition of 0.5 mM EBIO to the apical bath stimulated the control I_sc by 64% and reduced R_t by 21% (P < 0.05; paired data). Under Cl^–-free conditions, I_sc stimulation using EBIO was markedly attenuated. In the presence of an apical-to-basolateral K⁺ gradient and permeabilization of the apical membrane, the majority of the I_sc reflected the transcellular movement of K⁺ via basolateral K⁺ channels. Under these conditions, EBIO in combination with A23187 elicited nearly instantaneous 60–90% increases in I_sc that were sensitive to the calmodulin antagonist calmidazolium and the K⁺ channel blocker tetraethyl ammonium. In the presence of an apical-to-basolateral Cl^– gradient and nystatin permeabilization of the basolateral aspect, EBIO increased the Cl^–-dependent I_sc, an effect prevented by the channel blocker glibenclamide (0.3 mM). The latter compound also was used to determine the proportion of EBIO-evoked unidirectional ³⁶Cl^– fluxes in the presence of the Cl^– gradient that traversed the epithelium transcellularly. Overall, EBIO activated apical Cl^– channels and basolateral K⁺ channels (presumably those that are Ca²⁺ dependent), thereby suggesting that this compound, or related derivatives, may be suitable as topical agents to stimulate fluid transport across the tissue in individuals with lacrimal gland deficiencies. Ussing chamber; short-circuit current; electrolyte transport; chloride secretagogue; potassium conductance; 1-ethyl-2-benzimidazolinone; 1,10-phenanthroline     

Current-Voltage Curves of Single CI- Channels which Coexist with Two Types of K+ Channel in the Tonoplast of Chara corallina

A Cl^– channel and two types of K⁺ channel have been observed,by the use of the patch-clamp technique, in the membrane surroundingcytoplasmic droplets from Chara corallina. Measurements on cell-attachedpatches showed that the channel selective for Cl^– hada chord conductance of 21 pS at the resting membrane p.d. (mean= 11 mV, n = 19) and when open, passed an outward current of1.4 pA (n = 24 patches) at the resting p.d., with reversal ofthe direction of current at –54 mV (130 mol m^–3Cl^– in the external solution). The Cl^– concentrationin the cytoplasmic droplet calculated from the reversal p.d.was 15 mol m^–3. The channel strongly rectified outwardcurrent flow, but this rectification disappeared with symmetricalCl^– concentrations across detached patches of membrane.It is concluded that rectification observed in cell-attachedpatches is primarily due to asymmetric ^Cl– concentrationsrather than an asymmetry in energy barriers to Cl^– permeationin the channel or any voltage-dependent kinetics of the channel.The channel was rarely observed in detached patches despitebeing commonly observed in cell-attached patches. However, theabsence of Ca²⁺ at the cytoplasmic face of the membrane allowedobservation of the channel in detached patches for brief periods,during which ion substitution experiments revealed a permeabilitysequence of aspartate (76:33:1). A 100 pS K⁺ channel previously described by Luhring (1986) wasfrequently observed, in some instances simultaneously, witha channel having a conductance of 60 pS and displaying outwardrectification. This rectification was due to the channel remainingopen almost continuously for positive membrane potential differences(p.d.) and remaining shut almost continuously for negative p.d.'s.The 60 pS channel, like the 100 pS K⁺ channel, reversed currentflow at the resting p.d., suggesting that it was also permeableto K⁺. Key words: Plant ion-channels, chloride channel, potassium channel, patch-clamp     

Involvement of anion channel(s) in the modulation of the transient outward K(+) channel in rat ventricular myocytes

The cardiac Ca²⁺-independent transient outward K⁺ current (I_to), a major repolarizing ionic current, is markedly affected by Cl^– substitution and anion channel blockers. We reexplored the mechanism of the action of anions on I_to by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs⁺ substitution for intracellular K⁺. Replacement of most of the extracellular Cl^– with less permeant anions, aspartate (Asp^–) and glutamate (Glu^–), markedly suppressed the current. Removal of external Na⁺ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on I_to. In contrast, the permeant Cl^– substitute Br^– did not markedly affect the current, whereas F^– substitution for Cl^– induced a slight inhibition. The I_to elicited during Br^– substitution for Cl^– was also sensitive to blockade by 4-AP. The ability of Cl^– substitutes to induce rightward shifts of the steady-state inactivation curve of I_to was in the following sequence: NO₃^– > Cl^– Br^– > gluconate^– > Glu^– > Asp^–. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I_to similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp^– substitution for Cl^– was without significant effect on the intensity. These results suggest that the I_to channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated. transient outward potassium current; anion channel; actin cytoskeleton; myocyte; potassium ion