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     

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     

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca²⁺-permeable cation channels with subsequent entry of Ca²⁺ and stimulation of Ca²⁺-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl^– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca²⁺ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E₂ (PGE₂) formation, cation channel activity, Ca²⁺ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl^– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE_2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca²⁺ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes. annexin V; osmotic cell shrinkage; calcium; apoptosis     

Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl- channels in human osteoblasts

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl^– channels expressed in these cells as described previously. The reversal potential of the induced Cl^– current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl^–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl^– current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl^– channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling. potasium-chloride cotransporter; KCC1–KCC4; chloride channels; extracellular potassium concentration buffering     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Ionic mechanism for contractile response to hyposmotic challenge in canine basilar arteries

A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca²⁺ and by the voltage-dependent Ca²⁺ channel (VDCC) blocker nicardipine, but it was unaffected by thapsigargin, which depletes intracellular Ca²⁺ stores. The contraction was also inhibited by Gd³⁺ and ruthenium red, cation channel blockers, and Cl^– channel blockers DIDS and niflumic acid. The reduction of extracellular Cl^– concentrations enhanced the hypotonically induced contraction. Patch-clamp analysis showed that a hyposmotic challenge activated outwardly rectifying whole cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl^– concentration, indicating that the currents were carried by Cl^–. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca²⁺. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca²⁺ influx, thereby activating Ca²⁺-activated Cl^– channels. The subsequent membrane depolarization is likely to increase Ca²⁺ influx through VDCC and elicit contraction. stretch-activated cation channels; Ca²⁺-activated Cl^– channels; voltage-dependent Ca²⁺ channels; large-conductance Ca²⁺-activated K⁺ channels; gadolinium     

The effect of changes in ambient oxygen concentration on the bioelectric properties of middle ear mucosa

The purpose of the present study was to compare the effect of 24 h of exposure to 7% O₂ (normal middle ear physiological conditions) vs. 21% O₂ (found in the middle ear after ventilation tube placement) on transepithelial Na⁺ absorption and Cl^– secretion in cultured gerbil middle ear epithelial cell monolayers. Although no difference in apical Na⁺ absorption was identified, the UTP-induced stimulation of apical Cl^– secretion in the presence of apical Na⁺ channel blockade with amiloride was significantly enhanced after exposure to 21% O₂ compared with 7% O₂ exposure. In the presence of a calcium-activated Cl^– channel inhibitor, DIDS, UTP-induced stimulation of Cl^– secretion after 21% O₂ exposure was decreased, suggesting a role for calcium-activated Cl^– channels in middle ear Cl^– secretion in response to relative hyperoxia. ion channels; sodium; chloride; hypoxia     

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     

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl⁻ channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl⁻ channel in H₂O₂-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H₂O₂-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl⁻ channel can modulate H₂O₂-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.     

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     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

ClC-2 Activation Modulates Regulatory Volume Decrease

ClC-2 belongs to a large family of chloride channels and its expression in certain cell types is associated with the appearance of swelling-activated chloride (Cl⁻) currents. In the present report, we examined the hypothesis that ClC-2 plays a role in regulatory volume decrease by expressing ClC-2 in Sf9 cells using the baculovirus system. First, we showed that ClC-2 protein expression is associated with appearance of a Cl⁻ conductance which is activated by hypo-osmotic shock and can be distinguished from swelling-activated chloride currents endogenous to Sf9 cells on the basis of its pharmacology and specific inhibition by an anti-ClC-2 antibody. Second, we show that the rate of regulatory volume decrease is significantly enhanced in Sf9 cells expressing ClC-2 protein. Hence, our data support the hypothesis that ClC-2 is capable of mediating regulatory volume decrease. Received: 12 August/Revised: 23 October 1998     

Effects of Ions and Membrane Potential on the Elongation of the Unicellular Green Alga Closterium

Cells of the unicellular green alga Closterium ehrenbergii elongatedexclusively at septa and for 4–5 hours after cell division.Cell elongation was strongly inhibited by a decrease in eitherthe external concentration of Ca²⁺ or pH, and was also inhibitedby several competitive Ca²⁺ channel blockers. Changes in concentrationsof other external ions had no effect on the elongation. Theaverage concentrations of ions in the intracellular fluid ofthe interphase cell before cell division was as follows (inmM): K⁺=56.5, Na⁺=4.8, Ca²⁺=2.4, Mg²⁺=1.3, Cl^–=59.5; thepH was 7.4. The levels of K⁺, Na⁺ and Cl^– ions decreasedsignificantly with cell elongation, suggesting that this process,which proceeds with water uptake, surpasses ion absorption.The plasma membrane potential (Vm) in both the interphase cellsand in the elongating cells was in the range of –90 to–105 mV (interior negative). The Vm was entirely determinedby the simple diffusion of K⁺. A decrease in the external concentrationof Ca²⁺ caused depolarization, probably by an indirect effectof low Ca²⁺. Changes in the extracellular level of H⁺ and othercations barely affected Vm. Thus, external Ca²⁺ and H⁺ are concludedto affect cell elongation but not via a change in the Vm acrossthe plasma membrane. (Received February 29, 1988; Accepted June 8, 1988)     

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     

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.     

Protein Kinase A Activation Phosphorylates the Rat ClC-2 Cl− Channel but Does Not Change Activity

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl⁻ channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl⁻ currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl⁻ channel activity. Received: 20 November 2000/Revised: 28 March 2001     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat