Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents

Several proteins belonging to the ATP-binding cassettesuperfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1).We measured whole cell swelling-activatedCl currents(I_Cl,swell) inparental cells and cells expressing wild-type MDR1 or aphosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbolester reduced the rate of increase inI_Cl,swell only incells that express MDR1. PKC stimulation had no effect on steady-stateI_Cl,swell.Stimulation of protein kinase A (PKA) with 8-bromoadenosine3',5'-cyclic monophosphate reduced steady-state I_Cl,swell only inMDR1-expressing cells. PKA stimulation had no effect on the rate ofI_Cl,swellactivation. The effects of stimulation of PKA and PKC onI_Cl,swell wereadditive (i.e., decrease in the rate of activation and reduction insteady-stateI_Cl,swell). The effects of PKA and PKC stimulation were absent in cells expressing thephosphorylation-defective mutant. In summary, it is likely thatphosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl channels by independentmechanisms and that Ser-661, Ser-667, and Ser-671 are involved in theresponses ofI_Cl,swell tostimulation of PKA and PKC. These results support the notion that MDR1phosphorylation affectsI_Cl,swell.     

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.     

Cell volume regulation and swelling-activated chloride channels

Maintenance of a constant volume is essential for normal cell function. Following cell swelling, as a consequence of reduction of extracellular osmolarity or increase of intracellular content of osmolytes, animal cells are able to restore their original volume by activation of potassium and chloride conductances. The loss of these ions, followed passively by water, is responsible for the homeostatic response called regulatory volume decrease (RVD). Activation of a chloride conductance upon cell swelling is a key step in RVD. Several proteins have been proposed as candidates for this chloride conductance. The status of the field is reviewed, with particular emphasis on ClC-3, a member of the ClC family which has been recently proposed as the chloride channel involved in cell volume regulation.     

Proteolytic modification of swelling-activated Cl- current in LNCaP prostate cancer epithelial cells

The effects of intracellular application of trypsin on the Cl^– current induced by hypotonic cell swelling (I _Cl,swell) in human prostate cancer epithelial cells (LNCaP) was studied using the patch-clamp technique. In cells predialyzed with 1 mg/mL trypsin, I _Cl,swell developed and diminished in response to the application and withdrawal of hypotonic solution about three times faster than that in control cells. In trypsin-infused cells, I _Cl,swell also had about twofold higher current density and displayed considerably slowed voltage-dependent inactivation, which was quite pronounced in control cells at potentials above +60 mV. Trypsin-induced modification of I _Cl,swell could be prevented by coinfusion of 10 mg/mL soybean trypsin inhibitor, suggesting that proteolytic cleavage of essential intracellular structural domains of the I _Cl,swell-carrying volume-regulated anion channel (VRAC) was responsible for this functional modification. The effect of trypsin was not dependent on the presence of intracellular ATP. We conclude that VRACs, similarly to voltage-gated Na⁺, K⁺, and Cl^– channels, possess intracellular inactivation domain(s) subjected to proteolytic cleavage that may function in conformity with the classical ball-and-chain inactivation model.     

Regulation of swelling—activated chloride channels in embryonic chick heart cells

Swelling-activated Cl^- currents,I(Cl,swell),were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique.Genistein,an inhibitor of protein tyrosine kinase(PTK),suppressed I(Cl,swell).Under isosmotic condition phorbol 12-myristate 13-actetate(PMA),and activator of PKC,elicited the Cl^- current similar to that in hyposmotic solution,whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells,Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition,When the cells were treated with cytochalasin B(CB)or cytochalasin D(CD)under isosmotic condition the disruption of the F-actin integrity was observed,and I(C,l,swell). The results suggested that the role of PTK,probably receptor tyrosine kinase,for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin.Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl^- channels.These results demonstrate clearly that PTK,PKC and F-actin are important factors for regulation of I(Cl,swell),in embryonic chick heart cells as compared with often controversial results reported in different cell types.     

Swelling activated Cl- channels in microglia

Microglia have a swelling-activated Cl- current (which we call IClswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of IClswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I->Br->Cl- (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTP?S or GDP?S, or physiological levels of internal Mg2+. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. IClswell was reversibly inhibited by known Cl- channel blockers (NPPB, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na+/K+/Cl- symport inhibitor, bumetanide. Together, these results confirm the similarity of IClswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.     

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.     

Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases

Regulation of swelling-activated Cl(-) current (I(Cl,swell)) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I(Cl,swell) and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). I(Cl,swell) evoked by hyposmotic swelling was increased 181 +/- 17% by 100 microM genistein, and the genistein-induced current was blocked by the selective I(Cl,swell) blocker tamoxifen (10 microM). Block of Src with PP2 (10 microM) stimulated tamoxifen-sensitive I(Cl,swell) by 234 +/- 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 microM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I(Cl,swell) and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I(Cl,swell). Several lines of evidence argue that the PP2-stimulated current was I(Cl,swell): 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl(-) gradients, and 4) the current reversed near the Cl(-) equilibrium potential. To rule out contributions of other currents, Cd(2+) (0.2 mM) and Ba(2+) (1 mM) were added to the bath. Surprisingly, Cd(2+) suppressed the decay of I(Cl,swell), and Cd(2+) plus Ba(2+) eliminated time-dependent currents between -100 and +100 mV. Nevertheless, these divalent ions did not eliminate I(Cl,swell) or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I(Cl,swell), and regulation of I(Cl,swell) by the Src and EGFR kinase families of PTK is antagonistic.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Glibenclamide blocks volume-sensitive Cl- channels by dual mechanisms

To study the mechanisms of glibenclamide actions onvolume-sensitive Clchannels, whole cell patch-clamp studies were performed at various pHlevels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl currents in the entirerange of voltage examined (100 to +100 mV) and accelerated thedepolarization-induced inactivation at pH 7.5. When glibenclamide wasapplied from the intracellular side, in contrast, no effect wasobserved. At acidic pH, at which the weak acid glibenclamide existslargely in the uncharged form, the instantaneous current was, in avoltage-independent manner, suppressed by the extracellular drug atmicromolar concentrations without significantly affecting thedepolarization-induced inactivation. At alkaline pH, at which almostall of the drug is in the charged form, glibenclamide speeded theinactivation time course and induced a leftward shift of thesteady-state inactivation curve at much higher concentrations. Thus itis concluded that glibenclamide exerts inhibiting actions onswelling-activated Clchannels from the extracellular side and that the uncharged form ismainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.

     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.     

Potent block of Cl- channels by antiallergic drugs.

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p < 0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.     

Adenosine stimulates Cl- channels of nonpigmented ciliary epithelial cells

Ciliaryepithelial cells possess multiple purinergic receptors, and occupancyof A₁ andA₂ adenosine receptors isassociated with opposing effects on intraocular pressure. Aqueousadenosine produced increases in short-circuit current across rabbitciliary epithelium, blocked by removingCl and enhanced by aqueousBa²⁺. Adenosine's actions werefurther studied with nonpigmented ciliary epithelial (NPE) cells fromcontinuous human HCE and ODM lines and freshly dissected bovine cells.With gramicidin present, adenosine (3 µM) triggered isosmoticshrinkage of the human NPE cells, which was inhibited by theCl channel blockers5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 µM, the nonmetabolizable analog 2-chloroadenosine and AMP alsoproduced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine(1 µM) triggered increases of whole cell currents in HCE cells,which were partially reversible,Cl dependent, andreversibly inhibited by NPPB. Adenosine (10 µM) also stimulatedwhole cell currents in bovine NPE cells. We conclude that occupancy ofadenosine receptors stimulatesCl secretion in mammalianNPE cells.