Functionally additive fixed positive and negative charges in the CFTR channel pore control anion binding and conductance

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, a number of arginine and lysine residues have been shown to be important for Cl⁻ permeation. Among these, two in close proximity in the pore—Lys⁹⁵ and Arg¹³⁴—are indispensable for anion binding and high Cl⁻ conductance, suggesting that high positive charge density is required for pore function. Here we used mutagenesis and functional characterization to show that a nearby pore-lining negatively charged residue (Glu⁹²) plays a functionally additive role with these two positive charges. While neutralization of this negative charge had little effect on anion binding or Cl⁻ conductance, such neutralization was able to reverse the detrimental effects of removing the positive charge at either Lys⁹⁵ or Arg¹³⁴, as well as the similar effects of introducing a negative charge at a neighboring residue (Ser¹¹⁴¹). Furthermore, neutralization of Glu⁹² greatly increased the susceptibility of the channel to blockage by divalent S₂O₃²⁻ anions, mimicking the effect of introducing additional positive charge in this region; this effect was reversed by concurrent neutralization of either Lys⁹⁵ or Arg¹³⁴. Across a panel of mutant channels that introduced or removed fixed charges at these four positions, we found that many pore properties are dependent on the overall charge or charge density. We propose that the CFTR pore uses a combination of positively and negatively charged residues to optimize the anion binding and Cl⁻ conductance properties of the channel.     

Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl⁻ conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl⁻ conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO₂)₄²⁻ in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl⁻ channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl⁻ transport through a combination of failure to increase Cl⁻ conductance and increased susceptibility to channel block by cytosolic substances.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Multi-Ion Mechanism for Ion Permeation and Block in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

The mechanism of Cl⁻ ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl⁻ channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl⁻ concentration. Increasing extracellular Cl⁻ concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl⁻ and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl⁻ is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl⁻ concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl⁻ and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl⁻ permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl⁻ (but not gluconate) ions and the dependence of channel conductance on Cl⁻ concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN⁻) ions and anomalous mole fraction behavior seen in Cl⁻/SCN⁻ mixtures.     

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl⁻ and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl⁻ ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl⁻ ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl⁻ permeation, demonstrating their functional role in maximization of Cl⁻ flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl⁻. The location of these Cl⁻-attractive residues suggests that cytoplasmic Cl⁻ ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl⁻ ions from the cytoplasm.     

Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P_X/P_Cl) sequence NO₃ ⁻ > Cl⁻ > HCO₃ ⁻ > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P_X/P_Cl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Conserved Aromatic Residue Confers Cation Selectivity in Claudin-2 and Claudin-10b

In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr⁶⁷ mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na⁺ permeability, without affecting the Cl⁻ permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl⁻ permeability. The Y67F mutant restored the Na⁺ permeability, Cl⁻ permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore.     

Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl⁻ that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl⁻ conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl⁻ conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl⁻ binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl⁻. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Intracellular Proton-Transfer Mutants in a CLC Cl?/H+ Exchanger

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl⁻ and H⁺. Mutational analysis based on the known structure reveals several key residues required for coupling H⁺ to the stoichiometric countermovement of Cl⁻. E148 (Glu_ex) transfers protons between extracellular water and the protein interior, and E203 (Glu_in) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H⁺ transport while preserving Cl⁻ transport. We tested the role of Glu_in by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H⁺/Cl⁻ exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H⁺ coupling and severely inhibit Cl⁻ flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H⁺ transport with substantial Cl⁻ transport. Influence on H⁺ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu_in is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl⁻ movements to bring about Cl⁻/H⁺ exchange.     

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl⁻ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca²⁺]_i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl⁻ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I_sc) measurement in response to agonist-stimulated Cl⁻ secretion. FSK-stimulated Cl⁻ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca²⁺]_i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca²⁺]_i, activated Ras-related protein 2, and induced Cl⁻ secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I_sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl⁻ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl⁻ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl⁻>Br⁻>I⁻ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca²⁺]_i signaling pathway is involved in cAMP-stimulated Cl⁻ secretion, which is carried by a novel, previously undescribed Cl⁻ channel.     

Quantitative and Physiological Analyses of Chloride Dependence of Growth of Halobacillus halophilus

A quantitative analysis of the Cl⁻ dependence of growth of Halobacillus halophilus was performed. Optimal growth rates were obtained at Cl⁻ concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl⁻ concentration. Br⁻ but not I⁻, SO₄²⁻, NO₂⁻, SO₂⁻, OCN⁻, SCN⁻, BO₂⁻, or BrO₃⁻ could substitute for Cl⁻. To analyze the function of chloride, chloride concentration was determined. At low external Cl⁻ (Cl_e⁻) concentrations, the growth rate was low and Cl⁻ was excluded from the cytoplasm; increasing the Cl_e⁻ concentration led to an increase in the growth rate and an energy-dependent uptake of Cl⁻, thus decreasing the Cl_e⁻/internal Cl_i⁻ gradient from ≥10 at 0.1 M Cl_e⁻ to a nearly constant value of 2 at Cl_e⁻ concentrations which allowed optimal growth. Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl⁻-grown cultures are possible candidates for a chloride uptake system.     

Modulation of Voltage-dependent Properties of a Swelling-activated Cl? Current

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl⁻ current (I _Cl,swell) in BC₃H1 myoblasts. This Cl⁻ current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl⁻ currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg²⁺ concentration or reducing the Cl⁻ concentration of the extracellular solution. Replacing extracellular Cl⁻ by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN⁻ > I⁻ > NO₃ ⁻ > Cl⁻ >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg²⁺, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I _Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.     

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

Direct and Indirect Effects of Mutations at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO₂)₄²⁻ and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO₂)₄²⁻ ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO₂)₄²⁻. Inhibition by extracellular Pt(NO₂)₄²⁻ requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO₂)₄²⁻ is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO₂)₄²⁻ and extracellular Cl⁻ but not those between intracellular Pt(NO₂)₄²⁻ and extracellular Pt(NO₂)₄²⁻. We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.     

Interactions between permeant and blocking anions inside the CFTR chloride channel pore

Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is antagonized by extracellular Cl⁻. In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl⁻ (and other anions) and cytoplasmic Pt(NO₂)₄^2 − ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO₂)₄^2 − binding affinity also greatly strengthened antagonistic Cl⁻:blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO₂)₄^2 − ions but also with extracellular Cl⁻, and that altered blocker Cl⁻- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl⁻ binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl⁻ and Pt(NO₂)₄^2 − to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl⁻ binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl⁻ permeation, are discussed.     

The Epac1 Signaling Pathway Regulates Cl? Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

The apical membrane of intestinal epithelia expresses intermediate conductance K⁺ channel (KCNN4), which provides the driving force for Cl⁻ secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl⁻ secretion via stimulation of Rap2-phospholipase Cϵ-[Ca²⁺]_i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl⁻ secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl⁻ secretion and apical K⁺ conductance (I_K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′-O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K⁺ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I_K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I_K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.