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     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Inhibitors affecting the oxidizing side of Photosystem II at the Ca2+- and Cl−-sensitive sites

In addition to compounds which inhibit the function of calmodulin (a ubiquitous calcium regularity protein found in plants and mammalian tissues), Ca²⁺ or Cl⁻ channel blockers in mammalian tissues were also found to inhibit electron transport in Photosystem II submembrane preparations. Their inhibition was overcome by electron donation to P-680 by diphenylcarbazide (for all of the compounds used) and by H₂O₂ (except with trifluoperazine). Addition of Ca²⁺ and/or Cl⁻ also partially prevented the inhibitory action. We postulate that the inhibitory action occurs at the level of the water-splitting system at the site of Ca²⁺ modulation of the Cl⁻ cofactor requirement for O₂ evolution (as hypothesized in Nakatani, H.Y. (1984) Biochem. Biophys. Res. Comm. 120, 299–304).     

��5��1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K⁺ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin ligand, enhances BK channel current through both Ca²⁺- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca²⁺-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca²⁺ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca²⁺ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca²⁺ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997     

Niflumic Acid Affects Store-Operated Ca2+-Permeable (SOC) and Ca2+-Dependent K+ and Cl− Ion Channels and Induces Apoptosis in K562 Cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl^? and Ca²⁺-activated Cl^? (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8–10 %) apoptotic effect of endoplasmic reticulum Ca²⁺ mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca²⁺-activated K⁺ (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC₅₀ for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

A Ca2+-activated Cl− current in sheep parotid secretory cells

Previous studies have shown that the whole-cell current-voltage (I-V) relation of unstimulated sheep parotid cells is dominated by two K⁺ conductances, one outwardly and the other inwardly rectifying. We now show that once these K⁺ conductances are blocked by replacement of pipette K⁺ with Na⁺ and by the addition of 5 mmol/liter CsCl to the bath, there remains an outwardly rectifying conductance with a reversal potential of 0 mV. Replacement of 120 mmol/liter NaCl in the pipette solution with an equimolar amount of Na-glutamate shifted the reversal potential of this residual current to -55 mV, indicating that the conductance was Cl^? selective. The Cl^? current was activated by increasing the free Ca²⁺ in the pipette solution from 10 to 100 nmol/liter. When the Ca²⁺ concentration in the pipette solution was 10 nmol/liter, the relaxations observed in response to membrane depolarization could be fitted with a single exponential, whose time constant increased from 81 to 183 ms as the pipette potential was increased from -30 to +60 mV. Relaxation analysis showed that the current was activated by membrane depolarization. Reversal potential measurements in experiments in which external Cl^? was replaced with various anions, gave the following relative permeabilities: SCN^- (1.80) > I^- (1.09) > CI^- (1) > NO ₃ ^- (0.92) > Br^- (0.75). The relative conductances were: SCN^- (2.18) > I^- (1.07) > Cl^? (1.00) > Br^- (0.91) > NO ₃ ^- (0.50). The Cl^? current was blocked by NPPB (ID₅₀ ≈ 10 μm), DIDS (10 or 30 μmol/liter) and furosemide (100 μmol/liter).     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.     

Extracellular ATP activates both Ca2+- and cAMP-dependent Cl− conductances in rat epididymal cells

Activation of Ca²⁺ and cAMP-dependent Cl^– conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 m), activation of wholecell Cl^– current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl^– current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 m) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mm) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nm) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mm) or treatment of the cells with trifluoperazine (40 m), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl^– currents. No current activation by ATP was observed when cells were dialyzed with the IP₃ receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP₃-linked Ca²⁺-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca²⁺ and cAMP-dependent Cl^– conductances in epididymal cells.The authors wish to thank Mr. W.O. Fu for technical assistance. This work was supported by the Croucher Foundation, the University and Polytechnic Grants Committee.     

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.     

Identification and regulation of whole-cell Cl− and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca²⁺-activated K⁺ current was characterized by its voltage-dependent and Ca²⁺-dependent properties. When the extracellular K⁺ ion concentration was increased from 2 to 140 mm, the rereversal potential (E_k) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca²⁺-activated K⁺ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca²⁺-activated K⁺ channel studied with the single channel patch-clamp technique. The results suggest that the Ca²⁺-activated K⁺ current is the predominant, large conductance and Ca²⁺-dependent K⁺ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl^– current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl^– current showed an outward-rectifying pattern in symmetrical Cl^– solution. The Cl^– selectivity of the whole-cell current was confirmed by tail current analysis in different Cl^– concentration bath solutions. Several Cl^– channel blockers were found to be effective in blocking the outward-rectifying Cl^– current in MTAL cells. The cAMP-dependent Cl^– transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl^– sensitive dye using fluorescence microscopy. These results suggest that the Cl^– channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.     

Ca2+ Sensitivity of Synaptic Vesicle Dopamine, γ-Aminobutyric Acid,and Glutamate Transport Systems

The effect of Ca2⁺ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H⁺-ATPase activity, is strongly inhibited (70%) by 500 M Ca²⁺. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl^– concentrations, is not affected by Ca²⁺, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca²⁺ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 M Zn²⁺ that, like Ca²⁺, dissipates the proton concentration gradient by exchanging with H⁺. With respect to -aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca²⁺ or Zn²⁺ do not induce great alterations in the -aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca²⁺ or Zn²⁺ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H⁺ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.     

The Molecular Basis of Voltage-gated Ca2+ Channel Diversity: Is It Time for T?

The existence of diversity in the voltage activated Ca²⁺ channel populations of vertebrate cells has been long recognized. More recently, the molecular cloning of a considerable number of Ca²⁺ channel subunits from cDNA libraries has indicated that the range of possible Ca²⁺ channel phenotypes a cell can express may be even greater than was previously appreciated. A challenge of recent years has been to resolve how the properties of recombinant channels correspond with their counterparts experimentally characterized in native cells. In this short review I will outline the properties of both native and recombinant Ca²⁺ channels, and will then describe the current agreements and controversies concerning their relationships to each other. Received: 14 July 1997/Revised: 4 November 1997     

Volume-induced increase of K+ and Cl− permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+

Summary Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl^–, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca²⁺-activated K⁺ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca²⁺ ionophore A23187 in the presence of external Ca²⁺, and also by a sudden increase in external Ca²⁺. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca²⁺ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl^– permeability of the cell membrane there was no change in cell volume or Cl^– content when the K⁺ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca²⁺ ionophore A23187 in isotonic medium promoted Cl^– loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl^– permeability was increased in parallel with and even more than the K⁺ permeability. It is proposed that separate conductive K⁺ and Cl^– channels are activated during regulatory volume decrease by release of Ca²⁺ from internal stores, and that the effect is mediated by calmodulin.     

Efflux und Transport von Cl− und Rb+ in Maiswurzeln. Wirkung von Außenkonzentration,Ca++, EDTA und IES

Summary The efflux of ³⁶Cl and ⁸⁶Rb and the fluxes of these ions into the xylem were investigated using the device shown in Fig. 1.Efflux of ³⁶Cl is stimulated by external KCl while transport into the xylem is inhibited. Stimulation of the efflux appears to be stronger than inhibition of the transport.The stimulation of the efflux of ³⁶Cl was also observed with roots of intact seedlings.Assuming that the mode of transfer of Cl^– into the xylem (flux 3, Fig. 8) is diffusion exhibiting a linear isotherm (Luttge and Laties, 1966), these results suggest that the primary action of external salts is on the efflux across the plasma-lemma (Weigl, 1967, 1968). We were unable, however, to find a linear relationship between concentration and rate of chloride transport to the shoots of intact seedlings.With respect to the mode of ion transfer to the xylem (Weigl and Lüttge, 1965; Luttge and Laties, 1966) we have to be aware of the following facts:A linear isotherm cannot be taken to signify diffusive permeation (Torii and Laties, 1966; Luttge and Laties, 1966). If the Michaelis constant is extremely high relative to the ion concentration, the relationship between the ion concentration and the rate of a metabolic or mediated transport approaches linearity.The isotherm of the transport into the xylem may primarily reflect the difference of two large fluxes (4 and 5; Fig. 8).The transport data of Luttge and Laties (1966) need not be presented as a straight line (Fig. 6).If at high external ion concentrations the ratio of the ion concentration in the exudation sap to the external ion concentration approaches unity, diffusive permeation into the stele is still not proved to be the mode of migration, since at high stelar ion concentration flux 6 tends to become equal to flux 3.Considerations on radial ion transfer into the xylem depend on contemporary knowledge of the location of transport systems. Cl^–-uptake into root tips (2 mm) from solutions of 1–10 mM KCl did not exhibit a linear isotherm. These results are unpublished since the discrepancy to the results of Torii and Laties (1966) may be due to a higher content of vacuoles in our root tips. We feel it unlikely, however, that a linear isotherm of Cl^–-uptake into root tips is adequately explained by assuming that it is due to a lack of vacuoles while the sensibility to inhibitors is assumed to be due to the presence of vacuoles in root tips.Transport of Cl^– into the xylem is susceptible to inhibitors of oxydative phosphorylation, suggesting that this process, even at high external ion concentrations, is dependent on metabolic energy in contrast to the passive efflux from the cortical cells across the plasmalemma into the environment of the root. The precise location of the metabolic step(s) on the pathway of ions from the environment of the root to the xylem is unknown.The observed effects of Ca⁺⁺, EDTA and IAA may be considered in relation to the theory that auxin exerts its influence on growth by altering the diffusion potential across cell membranes (Brauner and Diemer, 1967). Growth is susceptible to the effect of Ca⁺⁺ and EDTA (Adamson, 1962; Setterfield, 1963; Thimann, 1963). Nevertheless, since IAA exerts no influence on ion fluxes in corn roots, it is not clear whether IAA really exerts its influence on growth by altering the diffusion potential across plant cell membranes. We might be dealing with occasional effects of secondary importance.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.