Current-voltage curve of electrogenic Cl− pump predicts voltage-dependent Cl− efflux inAcetabularia

Summary The current-voltage relationship of carrier-mediated, passive and active ion transport systems with one charge-carrying pathway can exactly be described by a simple reaction kinetic model. This model consists of two carrier states (one inside, one outside) and two pairs (forwards and backwards) of rate constants: a voltage-dependent one, describing the transport of charge and a voltage-insensitive one, summarizing all the other (voltage-independent) reactions. For the electrogenic Cl^– pump inAcetabularia these four rate constants have been determined from electrical measurements of the current-voltage relationship of the pump (Gradmann, Hansen & Slayman, 1981;in: Electrogenic Ion Pumps, Academic Press, New York). The unidirectional Cl^– efflux through the pump can also be calculated by the availiable reaction kinetic parameters.³⁶Cl^– efflux experiments on singleAcetabularia cells with simultaneous electrical stimulation (action potentials) and recording, demonstrate the unidirectional Cl^– efflux to depend on the membrane potential. After subtraction of an efflux portion which bypasses the pump, agreement is found between the measured flux-voltage relationship and the theoretical one as obtained from the reaction kinetic model and its parameters from the electrical data.     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Summary This paper provides the results of studies which characterized conductive³⁶Cl^– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive³⁶Cl^– uptake was studied under two different experimental conditions. In the first,³⁶Cl^– flux was driven by an inside positive voltage created with oppositely directed Cl^– and gluconate gradients. In the second, an inwardly direct K⁺ gradient was used to drive³⁶Cl^– uptake. By these two methods, voltage-sensitive³⁶Cl^– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total³⁶Cl^– flux. Separate paired studies demonstrated that the conductive³⁶Cl^– uptake was inhibited by the Cl^– channel blocker diphenylamine-2-carboxylate (DPC) with an IC₅₀ for DPC of 154 m. The voltagedependent³⁶Cl^– uptake had an activation energy of 6.4 kcal/mole. This³⁶Cl^– conductance had an anion selectivity sequence of I^–>Cl^–NO ₃ ^– gluconate.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

The energetics of Cl− active transport inChara

Summary The rate of Cl^– influx in intactChara was inhibited whenever the ATP concentration was reduced by application of metabolic inhibitors. In perfused cells, however, a net influx of Cl^– against its electrochemical gradient could be observed in the absence of ATP. Addition of ATP to the perfusion medium slightly stimulated Cl^– influx in one experiment but had no effect in another. Addition of ADP, NADH or metabolic inhibitors did not alter the influx rate. Consideration of the potential energy gradients across theChara plasmalemma in the perfused state leads to the conclusion that Cl^– influx occurs by cotransport with H⁺ or OH^–.     

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     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

PIKfyve-dependent regulation of the Cl− channel ClC-2

The widely expressed chloride channel ClC-2 is stimulated by the serum and glucocorticoid inducible kinase SGK1. The SGK1-dependent regulation of several carriers involves the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether SGK1-dependent regulation of ClC-2 similarly involves PIKfyve. The conductance of Xenopus oocytes is increased more than eightfold by ClC-2 expression. In ClC-2-expressing oocytes, but not in water-injected oocytes, the current was further enhanced by coexpression of either, PIKfyve or constitutively active ^S422DSGK1. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter the current in ClC-2-expressing oocytes and abrogated the stimulation of the current by PIKfyve-coexpression. The stimulating effect of PIKfyve was abolished by replacement of the serine with alanine in the SGK1 consensus sequence (^S318APIKfyve). Coexpression of ^S318APIKfyve significantly blunted the stimulating effect of ^S422DSGK1 on ClC-2-activity. In conclusion, PIKfyve is a potent stimulator of ClC-2-activity and contributes to SGK1-dependent regulation of ClC-2.     

Role of Cl− channels in primary brain tumour

There is tight interplay between Ca²⁺ and Cl⁻ flux that can influence brain tumour proliferation, migration and invasion. Glioma is the predominant malignant primary brain tumour, accounting for ˜80% of all cases. Voltage-gated Cl⁻ channel family (ClC) proteins and Cl⁻ intracellular channel (CLIC) proteins are drastically overexpressed in glioma, and are associated with enhanced cell proliferation, migration and invasion. Ca²⁺ also plays fundamental roles in the phenomenon. Ca²⁺-activated Cl⁻ channels (CaCC) such as TMEM16A and bestrophin-1 are involved in glioma formation and assist Ca²⁺ movement from intracellular stores to the plasma membrane. Additionally, the transient receptor protein (TRP) channel TRPC1 can induce activation of ClC-3 by increasing intracellular Ca²⁺concentrations and activating Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Therefore, Ca²⁺ and Cl⁻currents can concurrently mediate brain tumour cellular functions. Glioma also expresses volume regulated anion channels (VRACs), which are responsible for the swelling-induced Cl⁻ current, I_Cl,swell. This current enables glioma cells to perform regulatory volume decrease (RVD) as a survivability mechanism in response to hypoxic conditions within the tumour microenvironment. RVD can also be exploited by glioma for invasion and migration. Effective treatment for glioma is challenging, which can be in part due to prolonged chemotherapy leading to mutations in genes associated with multi-drug resistances (MRP1, Bcl-2, and ABC family). Thus, a potential therapeutic strategy for treatment of glioma can be through the inhibition of selected Cl⁻ channels.     

Reconstituted Cl− pump protein: A novel ion(Cl−)-motive ATPase

Cl^– absorption by theAplysia californica foregut is effected through an active Cl^– transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl^–-stimulated ATPase and ATP-dependent Cl^– transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg²⁺, Cl^–, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl^– accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl^– accumulation and electrical potential while an outwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl^– accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl^– accumulation, intraliposomal negative potential difference, and also Cl^–-stimulated ATPase activity. Both aspects of Cl^– pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl^–-ATPase actively transports Cl^– by an electrogenic process.     

Evidence for Cl− exchangers in perfused Carcinus gills

• 1.1. The effects of inhibitors and ion substitution on TBP (transbranchial potentials) and unidirectional ²²Na and ³⁶Cl fluxes from the bathing medium across the apical and basolateral sides into the perfusion solution J_a→b of isolated Carcinus gill epithelia were studied. Identical, diluted sea-water (DSW) and artificial solutions were used in the bathing solution and perfusate (202 ± 5 mM Na).
• 2.2. Externally applied bumetanide (10⁻³ M) did not affect ³⁶Cl and ²²Na fluxes. In addition, ³⁶Cl and ²²Na unidirectional J_a→b fluxes obtained by isosmotic substitution of co-ions did not provide evidence for the presence of a Na-K-2Cl co-transporter on the apical membrane.
• 3.3. 10⁻³ M 4-acetamido-4 isothiocyanostilbene 2,2 disulphonic acid (SITS) in the bathing solution and on both sides (apical and basolateral surfaces) effected a rapid, reversible and statistically significant inhibition of ³⁶Cl J_a→b, fluxes of 27.6 and 39.4%, respectively. In addition, 10 mM acetazolamide reduced Cl⁻ (external and both sides) and Na⁺ J_a→b, fluxes. Furthermore, Cl⁻ influxes were stimulated by addition of NaHCO₃ (15.5mM) on both epithelial sides by 64%.
• 4.4. On the basis of the experimental evidence described, it is suggested that there is a Cl⁻/HCO₃ (or OH⁻) antiporter located on the apical side of the gill surface.

     

Control of Cl− influx inChara by internal pH

Summary The possible regulation of Cl^– influx inChara by the cytoplasmic Cl^– concentration and cytoplasmic pH was investigated using both intact and intracellularly perfused cells. In perfused cells Cl^– influx was sensitive to changes in the internal Cl^– concentration but only when the concentration was less than 1mm.In intact cells the metabolic inhibitors, CCCP, DCMU, and oligomycin which inhibit Cl^– influx also reduced the cytoplasmic pH. A correlation between ATP concentration and cytoplasmic pH was shown to apply when the ATP concentration was lowered using these inhibitors. The possible relationships between ATP status, cytoplasmic pH, and Cl^– influx are discussed.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Cl− and F− anions regulate the architecture of protofibrils in fibrin gel

Ischemic heart disease is the leading cause of serious morbidity and mortality in Western society. One of the therapeutic approaches is based on the use of thrombolitic drugs that promote clot lysis. Even if the mechanisms leading to clot lysis are not completely understood, it is widely accepted that they depend on the complex biochemical reactions that occur among fibrin fibers and fibrinolitic agents, and by their ready diffusion into the fibers. Here we investigate the effects of specific anions on the architecture of protofibrils within fibrin fibers in fibrin gels prepared in a para-physiological solution. The results obtained through small-angle X-ray scattering (SAXS) demonstrate that the characteristic axial and longitudinal repeat distances among protofibrils are strongly affected by the action of Cl⁻ and F⁻ anions.     

Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na⁺ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl^– conductance and we show that the sizes of both conductances vary with the Cl^– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl^– concentration rises from 5 to 150 mmol/liter, the size of the inward Na⁺ current declines, the decline being half-maximal when the Cl^– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl^– concentration increases, the inward Cl^– current remains at a constant low level until the Cl^– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl^– in the pipette solution was replaced by other anions indicate that the Na⁺ current is suppressed by intracellular Br^-, Cl^– and NO ₃ ^- but not by intracellular I^-, glutamate or gluconate. Our studies also show that the Cl^– conductance allows passage of Cl^– and Br^- equally well, I-less well, and NO ₃ ^- , glutamate and gluconate poorly, if at all. The findings with NO ₃ ^- are of particular interest because they show that suppression of the Na⁺ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl^– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na⁺ and Cl^– conductances by the cytosolic Cl^– concentration. Since the cytosolic Cl^– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na⁺ and Cl^– conductances may provide a mechanism by which ductal Na⁺ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements.