Anion Selectivity Sequence Research Articles

Anion-π catalysis: bicyclic products with four contiguous stereogenic centers from otherwise elusive diastereospecific domino reactions on π-acidic surfaces.

Anion-π interactions have been introduced recently to catalysis. The idea of stabilizing anionic intermediates and transition states on π-acidic surfaces is a new fundamental concept. By now, examples exist for asymmetric enolate, enamine, iminium and transamination chemistry, and the first anion-π enzyme has been created. Delocalized over large aromatic planes, anion-π interactions appear particularly attractive to stabilize extensive long-distance charge displacements during domino processes. Moving on from the formation of cyclohexane rings with five stereogenic centers in one step on a π-acidic surface, we here focus on asymmetric anion-π catalysis of domino reactions that afford bicyclic products with quaternary stereogenic centers. Catalyst screening includes a newly synthesized, better performing anion-π version of classical organocatalysts from cinchona alkaloids, and anion-π enzymes. We find stereoselectivities that are clearly better than the best ones reported with conventional catalysts, culminating in unprecedented diastereospecificity. Moreover, we describe achiral salts as supramolecular chirality enhancers and report the first artificial enzyme that operates in neutral water with anion-π interactions, i.e., interactions that are essentially new to enzymes. Evidence in support of contributions of anion-π interactions to asymmetric catalysis include increasing diastereo- and enantioselectivity with increasing rates, i.e., asymmetric transition-state stabilization in the presence of π-acidic surfaces and inhibition with the anion selectivity sequence NO3- > Br- > BF4- > PF6-.

Functional and transport analyses of CLCN5 genetic changes identified in Dent disease patients.

Dent disease type 1, an X‐linked inherited kidney disease is caused by mutations in electrogenic Cl−/H+ exchanger, ClC‐5. We functionally studied the most frequent mutation (S244L) and two mutations recently identified in RKSC patients, Q629X and R345W. We also studied T657S, which has a high minor‐allele frequency (0.23%) in the African‐American population, was published previously as pathogenic to cause Dent disease. The transport properties of CLC‐5 were electrophysiologically characterized. WT and ClC‐5 mutant currents were inhibited by pH 5.5, but not affected by an alkaline extracellular solution (pH 8.5). The T657S and R345W mutations showed the same anion selectivity sequence as WT ClC‐5 (SCN −>NO3−≈Cl−>Br−>I−). However, the S244L and Q629X mutations abolished this anion conductance sequence. Cell surface CLC‐5 expression was quantified using extracellular HA‐tagged CLC‐5 and a chemiluminescent immunoassay. Cellular localization of eGFP‐tagged CLC‐5 proteins was also examined in HEK293 cells with organelle‐specific fluorescent probes. Functional defects of R345W and Q629X mutations were caused by the trafficking of the protein to the plasma membrane since proteins were mostly retained in the endoplasmic reticulum, and mutations showed positive correlations between surface expression and transport function. In contrast, although the S244L transport function was significantly lower than WT, cell surface, early endosome, and endoplasmic reticulum expression was equal to that of WT CLC‐5. Function and trafficking of T657S was equivalent to the WT CLC‐5 suggesting this is a benign variant rather than pathogenic. These studies demonstrate the useful information that can be gained by detailed functional studies of mutations predicted to be pathogenic.

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-β-D-glucosamine-Based Artificial Ion-Transport Systems.

Cyclo-oligo-(1→6)-β-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed.

Functional and Transport Analysis of CLCN5 Mutations Found in Dent Disease Patients

Dent disease, an X-linked kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. In published Dent patients, S244L is the most frequently found mutation. Q629X and R345W are two novel mutations diagnosed by the Mayo Clinic RKSC. T657S was reported as pathogenic in a Dent disease patient and high minor-allele frequency (0.391%) in the African-American population without any pathophysiology data. The transport properties of CLC-5 were characterized by electrophysiology experiments showing rectified outward currents at positive holding voltages in the presence of 104 mM Cl- while removal of chloride abolished the currents. Currents of WT and ClC-5 mutants were inhibited by extracellular pH at 5.5 but not pH8.5. The T657S and R345W showed same anion selectivity sequence as WT (SCN->NO3-∼Cl->Br->I-). However, the S244L and Q629X lost the anion conductance sequence. A HA tag, shown not to interfere CLC-5 transport function, was added to the extracellular loop of the CLC-5 protein enabling chemiluminescence method to quantify ClC-5 surface expression. Oocytes expressing T657S, R345W, and Q629X mutations showed positive correlations between transport function and cell membrane protein expression indicating protein trafficking problem. In contrast, S244L function was significantly lower than WT CLC-5 whereas protein expression did not significantly differ from the HA tagged CLC-5 WT suggesting defected transport function. The function and trafficking of T657S was equivalent to the CLC-5 WT and seems to be a benign mutation. DK101405, AHA-SDG2640146

TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

TMEM16F is a component of a Ca2+-activated Cl− channel but not a volume-sensitive outwardly rectifying Cl− channel

TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl(-) channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca(2+) ionophore, ionomycin, or by an increase in the intracellular free Ca(2+) concentration. The free Ca(2+) concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca(2+) level, in contrast to TMEM16A/B currents. The Ca(2+)-activated TMEM16F currents were anion selective, because replacing Cl(-) with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I(-) > Br(-) > Cl(-) > F(-) > aspartate(-). Niflumic acid, a Ca(2+)-activated Cl(-) channel blocker, inhibited the TMEM16F-dependent Cl(-) currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl(-) channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca(2+)-activated Cl(-) channel with a Ca(2+) sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

The Role of Swelling-Activated Chloride Currents (ICL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Background/Aims: Articular chondrocytes dwell in an environment that is continuously changing its osmolarity as a consequence of mechanical loading, yet their volume regulation capabilities (RVD) are not fully understood. This work aimed to determine the osmotic sensitivity of freshly isolated rat chondrocytes, their RVD capabilities and to study the properties of any anion currents associated. Methods: Cell volume responses were determined by microscopy. Whole cell patch clamp was used to record ion currents. Results: Chondrocytes showed to be osmotically sensitive and capable of RVD in a size-dependent manner. RVD was accompanied by activation of outwardly rectifying chloride currents, featuring time and voltage independent activation and inactivation at most depolarizing voltage levels, with an anion selectivity sequence of: SCN->I-≅NO3->Br->Cl->F->> Gluconate-> Methanesulphonate-, corresponding to Eisenman’s sequence I. They were also permeable to taurine. These currents were blocked by DIDS, SITS, 9-AC and NPPB and by drugs that block ICl,swell such as fluoxetine, phloretin, DCPIB and tamoxifen. RT-PCR assays show the presence of mRNA for CLC-3 and TMEM16A, that had been proposed as molecular determinants of ICl,swell currents. Conclusions: These findings indicate that freshly isolated rat articular chondrocytes have ICl,swell. The further finding that ICl,swell blockers alter the RVD response suggest that ICl,swell is involved in the RVD response of rat articular chondrocytes.

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl(-) channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na(+) conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl(-) current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl(-) current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn(2+) sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn(2+) (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl(-) channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na(+) transport in this surface epithelium under basal physiological conditions.

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.

Characterization of Glial Cell K-Cl Cotransport

Background: The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells. Methods: We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl^--dependent bumetanide-insensitive Rb⁺ (a K⁺ congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb⁺ influx was measured at a fixed external Cl concentrations, [Cl^-]_e, as a function of varying external Rb concentrations, [Rb⁺]_e, and at a fixed [Rb⁺]_e as a function of varying [Cl^-]_e, and with equimolar Cl replacement by anions of the chaotropic series. Results: RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K_m for KCC-mediated Rb⁺ influx was 15 mM [Rb⁺]_e, and V_max 12.5 nmol Rb⁺ * mg protein^-1 * minute^-1. The calculated apparent K_m for external Cl^- was 13 mM and V_max 14.4 nmol Rb⁺ * mg protein^-1 * minute^-1. The anion selectivity sequence of the furosemide-sensitive Rb⁺ influx was Cl^->>Br-=NO₃^->I^-=SCN^->>Sfm^- (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb⁺ influx. A ñ50% NEM-induced loss of intracellular K⁺ was prevented by furosemide. Conclusion: We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.

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 Gd(3+), Cd(2+), and Zn(2+), 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.

Predominant constitutive CFTR conductance in small airways

BackgroundThe pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter.MethodsWe introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole.ResultsIn bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath) of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25), but when gluconate replaced luminal Cl-, the bionic Cl- diffusion potentials (-58 ± 3 mV; n = 25) were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTRInh-172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways.ConclusionThese results indicate that the small airway of the pig is characterized by a constitutively active Cl- conductance that is most likely due to CFTR.

Cl−Influx into Rat Cortical Lens Fiber Cells Is Mediated by a Cl−Conductance That Is Not ClC-2 or -3

Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible. Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations. Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature. Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.

Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression.

We examined the effects of intracellular Cl- concentration ([Cl-]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71-85, 2002). Treatment with cAMP (100 microM 8-cpt-cAMP plus 100 microM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl- secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in alpha-toxin-permeabilized monolayers to have the anion selectivity sequence NO3- > Br- > Cl- > I-. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS-Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl-] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl-] in the apical solution. This inhibitory effect of [Cl-]i occurred regardless of the presence or absence of net Cl- transport. However, from fluorometric measurements using the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl- secretion, produced a decrease of [Cl-]i from 76 +/- 14 to 36 +/- 8 mM (P = 0.03). Thus it might be expected that activation of Cl- secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl-]i from 15 to 145 mM decreased AS-Isc from 24.5 +/- 1.0 to 10.2 +/- 1.6 microA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 +/- 0.16 to 1.32 +/- 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 +/- 1.2 to 8.2 +/- 1.4 microA/fmol.

Expression of novel isoforms of the CIC-1 chloride channel in astrocytic glial cells in vitro.

Chloride channels play an important role in glial astrocyte function. However, in astrocytes, no chloride channels besides the gamma-aminobutyric acid (GABA)A receptor, glycine receptor, and ClC-2 chloride channels have been molecularly identified. In this study, we examined the expression of the ClC-1 chloride channel in rat astrocytic glioma C6 cells and rat primary astrocytes. Five isoforms of ClC-1, but not skeletal muscle ClC-1 (SM ClC-1), were found to be expressed in C6 cells. Comparison with rat SM ClC-1 showed that common features shared by these isoforms are a short 3' end with a deletion of the nucleotides from 3115 to 3197 and a substitution of T by C at nucleotides 480 and 1733. Three of the five isoforms, M1, M2, and M3, were produced by partial deletion of ClC-1 exon 7, partial insertion of ClC-1 exon 7a, and a TAG insertion at nucleotide 858, respectively. One of the two remaining isoforms, M4, was produced by partial deletion of ClC-1 exon 8 at nucleotide 937; the other, M5, was the same as SM ClC-1 except for the short 3' end and substitutions at the two positions. Only the M5 isoform could be expressed as a functional channel in Xenopus oocytes. This glial isoform exhibited less dependence on voltage and extracellular Cl- than rat SM ClC-1. However, the anion selectivity sequence and the anthracene-9-carboxylic acid (9-AC) sensitivity of this channel were the same as for SM ClC-1. Since whole-cell recordings failed to detect ClC-1-like Cl- currents in C6 cells, it appears that the ClC-1 isoform is functioning in intracellular organelles. In rat primary astrocytes, we found that the M2 isoform as well as two additional distinct isoforms were expressed. The present study showed that astrocytic glial cells express multiple isoforms of the ClC-1 chloride channel, which has been thought to be expressed almost exclusively in the skeletal muscle.

A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel.

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl− cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl− transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl− channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10−5 M forskolin, but the channel was inhibited by phorbol ester (10−6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NP o increased threefold), or pH increased from 6.4 to 8.0 (NP o increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl− relative permeability was 0.09, and the anion selectivity sequence Cl− ∼ I−> Br− ∼ NO3 − > F−. Intracellular NPPB (10−4 M) and DPC (10−3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl− channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.

ATP regulation of epithelial Cl- channels - new challenges?

ATP regulation of epithelial Cl- channels - new challenges?

ATP regulation of epithelial Cl- channels--new challenges?

ATP regulation of epithelial Cl- channels--new challenges?

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes.

The materno-fetal transfer of metabolites and nutrients requires the operation of specific transport mechanisms through syncytiotrophoblast membranes. Electrophysiological studies on these cells are scarce and, because of their syncytial nature, whole-cell current recordings have not been carried out. We have now studied whether or not ion channels from the human syncytiotrophoblast microvillous (hSM) membrane can be transplanted to Xenopus oocytes. Sixty-two percent of hSM-injected oocytes displayed lower resting potential and higher membrane conductance than uninjected cells. The increased membrane conductance was due to the incorporation of Cl(-) channels, because neither replacing Na(+) in the bathing solution by N-methyl- D-glucamine or K(+), nor withdrawing Ca(2+) had any significant effect on the currents elicited by voltage pulses. In contrast, substitution of Cl(-) by different anions markedly affected the membrane conductance, giving an anion selectivity sequence of I(-)>Br(-)>Cl(-)>methanosulfonate congruent with gluconate. In addition, disulfonic stilbenes and gluconate, but not anthracene-9-carboxylic acid, blocked the transplanted channels. These properties are compatible with those of placental Cl(-) "maxi" channels. It is concluded that functional Cl(-) channels from the hSM become effectively incorporated into the Xenopus oocyte membrane, where their function can be studied in detail.

Na+-alanine uptake activates a Cl- conductance in frog renal proximal tubule cells via nonconventional PKC.

Hyposmotically induced swelling of frog renal proximal tubule cells activates a DIDS-sensitive, outwardly rectifying Cl- conductance via a conventional protein kinase C (PKC). This study examines whether Na+-alanine cotransport similarly activates a DIDS-sensitive Cl- conductance in frog renal proximal tubule cells. On stimulation of Na+-alanine cotransport, the DIDS-sensitive current (I(DIDS-Ala)) increased markedly over time. I(DIDS-Ala) exhibited outward rectification, a Na+/Cl- selectivity ratio of 0.19 +/- 0.03, and an anion selectivity sequence Br- = Cl- > I- > gluconate-. Activation of I(DIDS-Ala) was dependent on ATP hydrolysis and PKC-mediated phosphorylation and was inhibited by hyperosmotic conditions. Activation could be not ascribed to a conventional PKC isoform, as I(DIDS-Ala) was not affected by removing Ca2+ or by phorbol ester treatment, suggesting a role for a nonconventional PKC isoform, either novel or atypical. We conclude that Na+-alanine cotransport activates a DIDS-sensitive Cl- conductance via a nonconventional PKC isoform. This contrasts with the hyposmotically activated Cl- conductance, which requires conventional PKC activation.