Intracellular regulation of human ClC‐5 by adenine nucleotides

ClC-5, an endosomal Cl⁻/H⁺ antiporter that is mutated in Dent disease, is essential for endosomal acidification and re-uptake of small molecular weight proteins in the renal proximal tubule. Eukaryotic chloride channels (CLCs) contain two cytoplasmic CBS domains, motifs present in different proteins, the function of which is still poorly understood. Structural studies have shown that ClC-5 can bind to ATP at the interface between the CBS domains, but so far the potential functional consequences of nucleotide binding to ClC-5 have not been investigated. Here, we show that the direct application of ATP, ADP and AMP in inside-out patch experiments potentiates the current mediated by ClC-5 with similar affinities. The nucleotides increase the probability of ClC-5 to be in an active, transporting state. The residues Tyr 617 and Asp 727, but not Ser 618, are crucial for the potentiation. These results provide a mechanistic and structural framework for the interpretation of nucleotide regulation of a CLC transporter.     

Mechanism of Interaction of Niflumic Acid with Heterologously Expressed Kidney CLC-K Chloride Channels

CLC-K Cl^- channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter’s syndrome, and mutations in the associated subunit barttin produce Bartter’s syndrome and deafness. We have previously found that 3-phenyl-CPP blocks hClC-Ka and rClC-K1 from the extracellular side in the pore entrance. Recently, we have shown that niflumic acid (NFA), a nonsteroidal anti-inflammatory fenamate, produces biphasic behavior on human CLC-K channels that suggests the presence of two functionally different binding sites: an activating site and a blocking site. Here, we investigate in more detail the interaction of NFA on CLC-K channels. Mutants that altered block by 3-phenyl-2-(p-chlorophenoxy)propionic acid (CPP) had no effect on NFA block, indicating that the inhibition binding site of NFA is different from that of 3-phenyl-CPP and flufenamic acid. Moreover, NFA does not compete with extracellular Cl^- ions, suggesting that the binding sites of NFA are not located deep in the pore. Differently from ClC-Ka, on the rat homologue ClC-K1, NFA has only an inhibitory effect. We developed a quantitative model to describe the complex action of NFA on ClC-Ka. The model predicts that ClC-Ka possesses two NFA binding sites: when only one site is occupied, NFA increases ClC-Ka currents, whereas the occupation of both binding sites leads to channel block.     

Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life

During fetal development, the lung is filled with fluid that is secreted by an active Cl- transport promoting lung growth. The basolateral Na⁺,K⁺,2Cl^- cotransporter (NKCC1) participates in Cl^- secretion. The apical Cl^- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na⁺ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl^- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl^- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl^- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Cl^- channels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition.     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl^- with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl^- cotransporter NKCC1 and the K⁺/Cl^- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.     

Intracellular Proton Access in a Cl?/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H⁺-gated Cl⁻ channels and Cl⁻/H⁺ antiporters. Transmembrane H⁺ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl⁻ ion pathway through these proteins, but the H⁺ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H⁺ in its path through the protein. The extracellular-facing H⁺ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu_in, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu_in. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl⁻/H⁺ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu_in physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu_in.     

GlialCAM,a CLC-2 Cl Channel Subunit,Activates the Slow Gate of CLC Chloride Channels

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl^- channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.     

The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0

Transmembrane proton transport is of fundamental importance for life. The list of H⁺ transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl⁻/H⁺ antiporters. Other CLC proteins are instead passive Cl⁻ selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl⁻/H⁺ antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a “fast gate”. A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate’s pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH⁻ anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl⁻ ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate’s pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.     

Substrate‐driven conformational changes in ClC‐ec1 observed by fluorine NMR

The CLC ‘Cl⁻ channel'' family consists of both Cl⁻/H⁺ antiporters and Cl⁻ channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl⁻ permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent ¹⁹F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl⁻ permeation pathway and show the usefulness of ¹⁹F NMR for studying conformational changes in membrane proteins of known structure.     

GlialCAM,a CLC-2 Cl- Channel Subunit,Activates the Slow Gate of CLC Chloride Channels

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl^- channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.     

A model for the mechanism of chloride activation of oxygen evolution in photosystem II

A hypothesis is proposed to explain the function of Cl^- in activating the oxygenevolving complex (OEC) of photosystem II (PS II), based on the results of recent ³⁵Cl-NMR studies. The putative mechanism involves Cl^- binding to two types of sites. An intrinsic site is suggested to be composed of three histidyl residues (His 332 and His 337 from D1 and His 337 D2). It is proposed that Cl^- binding to this site accelerates the abstraction of H⁺ from water by raising the pK_a's of the histidine imidazole groups. Cl^- binding also stimulates the transfer of H⁺ from this intrinsic site to a set of extrinsic sites on the 33 kD extrinsic polypeptide. The extrinsic Cl^- binding sites are suggested to involve four protein domains that are linked together by salt-bridge contacts. Chloride and H⁺ donated from the intrinsic site attack these intramolecular salt-bridges in a defined sequence, thereby exposing previously inaccessible Cl^- and H⁺ binding sites and stimulating the oxidation of water. This hypothesis also proposes a possible structure for the Mn active site within the D1/D2 complex. Specific amino-acid residues that are likely to participate as Mn lignads are identified on the lumenal portions of the D1 and D2 proteins that are different from those in the L and M subunits of photosynthetic bacteria; the choice of these residues is based on the metal coordination chemistry of these residues, their location within the polypeptide chain, the regularity of their spacing, and their conservation through evolution. The catalytic Mn-binding residues are suggested to be D-61, E-65, E-92, E-98, D-103; D-308, E-329, E-342 and E-333 in D1, and H-62, E-70, H-88, E-97, D-101; E-313, D-334, E-338 and E-345 in D2. Finally, this hypothesis identifies sites on both D2 and the 33 kD extrinsic polypeptide that might be involved in high- and low-affinity Ca²⁺ binding.To whom correspondence should be addressed     

The effect of chloride on the thermal inactivation of oxygen evolution

The effect of Cl depletion on the sensitivity of the oxygen-evolving complex of Photosystem II (PS II) to heat treatment was examined by a parallel study of the Hill activity (H₂O2,6-dichlorophenolindophenol), Cl^- binding (by ³⁵Cl-NMR) and Mn release (by EPR). The extent of thermal inactivation in spinach thylakoids was found to depend on the degree of Cl^- depletion in the sample. In partially Cl^--depleted thylakoids, mild heating (38°C, 3 min) was found to eliminate inflections in plots of both Hill activity versus [Cl^-] (at low light intensity) and excess ³⁵Cl-NMR linewidth versus [Cl^-] (in the dark). In PS II membranes, the same treatment reduced the differences between the linewidth maxima and minima, particularly in the region of 0.3 mM and 7.0 mM Cl^-, as compared to unheated membranes. These results indicate that mild heating affects the Cl^--binding domains within the oxygen-evolving complex, OEC, EPR measurements of the temperature dependence of Mn release from heated thylakoids show that Mn release begins to correlate with the loss of Hill activity only at higher temperatures, where the OEC is already substantially inactivated. We conclude from these studies that the Cl^--binding domains of the OEC constitute a principal site of damage by heat treatment.     

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl^- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl^- from proteoliposomes following the addition of the K⁺ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H⁺/Cl^- exchanger, as a model system. The efflux assay can be utilized to study any Cl^- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.     

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl^- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl^- channels. The two major populations constituted Ohmic Cl^- channels with limiting conductance (γ_125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl^- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10^-14, 5.7 (±0.7) × 10^-14, and 32 (±2) × 10^-14 cm³/sec, for the 10, 30 and 150 pS Cl^- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl^- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E _Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl^- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl^- channels are organized in clusters. Received: 10 October 1996/Revised: 8 January 1997     

Mechanisms of block of muscle type CLC chloride channels (Review)

CLC proteins are a nine-member gene family of Cl- channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a breakthrough for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.     

Modulation of the slow/common gating of CLC channels by intracellular cadmium

Members of the CLC family of Cl⁻ channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl⁻ channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd²⁺ reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd²⁺-coordinating residues. The inhibition of the current of CLC-0 by Cd²⁺ was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd²⁺-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd²⁺-binding site. Kinetic studies showed that Cd²⁺ inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd²⁺ modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl⁻ channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd²⁺ likely depends on their alteration of subunit interactions.     

Investigating CFTR and KCa3.1 Protein/Protein Interactions

In epithelia, Cl^- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl^- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl^- ions and electrolytes needs however to be coupled to an increase in K⁺ conductance in order to recycle K⁺ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K⁺ efflux is ensured by K⁺ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca²⁺ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca²⁺.     

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl/H Antiporters

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl⁻] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.     

Ion transport in primary cultures from human sweat gland coils studied with X-ray microanalysis

Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular Cl concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23187. The muscarinic agonists also lowered the cellular K concentration. Cl^- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Cl^- secretion only from a subpopulation of the cells. Also ATP stimulated Cl^- secretion, indicating the presence of purinergic receptors. The results suggest that some of the cells in addition to Ca²⁺ -regulated Cl^- channels also possess cAMP-activated Cl^- channels. Hence, the primary cultures still possess the Cl^- transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.