Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γ_j, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor D_x/D_o, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.     

Mechanisms of Anion and Cation Permeations in the Resting Membrane of a Barnacle Muscle Fiber

The resting membrane of a barnacle muscle fiber is mostly permeable to cations in a solution of pH 7.7 whereas it becomes primarily permeable to anions if the pH is below 4.0. Mechanisms of ion permeation for various monovalent cations and anions were investigated at pH 7.7 and 3.9, respectively. Permeability ratios were obtained from the relationship between the membrane potential and the concentration of the test ions, and ionic conductances from current-voltage relations of the membrane. The permeability sequence for anions (SCN > I > NO₃ > Br > ClO₃ > Cl > BrO₃ > IO₃) was different from the conductance sequence for anions (Br, Cl > ClO₃, NO₃ > SCN). In contrast, the permeability and conductance sequences were identical for cations (K > Rb > Cs > Na > Li). The results suggest that anion permeation is governed by membrane charges while cation permeation is via some electrically neutral mechanism.     

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.     

Anion channels in the sea urchin sperm plasma membrane

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO₃^? > CNS^? > Br^? > CI^?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.     

Anion permeation in Ca(2+)-activated Cl(-) channels

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.     

The Ca-activated chloride channel of Ascaris suum conducts volatile fatty acids produced by anaerobic respiration: A patch-clamp study

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of carboxylic acids, a number of which are products of anaerobic respiration, was investigated using the patch-clamp technique and isolated inside-out patches of muscle membrane. The channel has a conductance of 140 pS in symmetrical 140 mm chloride. Replacement of internal chloride with various carboxylic acids (140 mm) caused large hyperpolarizing shifts in the reversal potential. Permeability ratios, relative to chloride, were calculated for each acid. The relationship between permeability ratio and ionic size is inverse and linear predicting a pore diameter of 6.55 Å. This simple relationship was not observed between ionic size and conductance. Calculation of the transition state energy required to transfer a single methyl group from aqueous phase to the binding site afforded a value that was low but favorable, indicating a cationic binding site of low field strength. As the channel is able to open fully at the resting membrane potential of Ascaris and is permeable to fatty acids produced by anaerobic respiration, the possible role of this channel in the removal of metabolic products across the muscle membrane is discussed.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.). M. Valkanov was sponsored by The British Council.     

Theoretical studies on identity SN2 reactions of lithium halide and methyl halide: A microhydration model

Reactions of lithium halide (LiX, X = F, Cl, Br and I) and methyl halide (CH₃X, X = F, Cl, Br and I) have been investigated at the B3LYP/6-31G(d) level of theory using the microhydration model. Beginning with hydrated lithium ion, four or two water molecules have been conveniently introduced to these aqueous-phase halogen-exchange S_N2 reactions. These water molecules coordinated with the center metal lithium ion, and also interacted with entering and leaving halogen anion via hydrogen bond in complexes and transition state, which to some extent compensated hydration of halogen anion. At 298 K the reaction profiles all involve central barriers ΔE _cent which are found to decrease in the order F > Cl > Br > I. The same trend is also found for the overall barriers (ΔE _ovr ) of the title reaction. In the S_N2 reaction of sodium iodide and methyl iodide, the activation energy agrees well with the aqueous conductometric investigation.     

Anions permeate and gate GCAC1, a voltage-dependent guard cell anion channel

GCAC1 is a strongly voltage-dependent anion channel in the guard-cell plasma membrane of Vicia faba . In patch–clamp experiments, we have investigated the permeation and gating properties of GCAC1 with respect to its anion dependence in the whole-cell and excised-patch configuration. The relative permeability followed the order SCN^– > NO₃^– > Br^– > Cl^–, while the single-channel conductances in symmetrical anionic solutions exhibited a nearly inverse sequence. The Cl^– dependence of inward currents (Cl^– release) is characterized by a maximum single-channel conductance of 89 pS half-saturating at 87 mM cytoplasmic chloride. In addition to this substrate saturation, anion release was also dependent on the external Cl^– activity ( K _m = 16 mM). In the presence of SCN^– and Cl^–, the single-channel conductance exhibited an anomalous mole-fraction dependence, identifying GCAC1 as a multi-ion single-file pore. Using anions with increasing ionic size, a minimum pore diameter of 0.5 nm was assumed from their relative permeabilities. In line with an anion-selective channel, a tenfold increase in the extracellular anion activity shifted the reversal potential by –59.8 mV. Simultaneously, the half-activation potential shifted negatively by about 23 mV. A further analysis of the anion dependence revealed that extracellular rather than cytosolic anions affect the gating process of GCAC1. From anion substitution experiments, we conclude that anion concentration and species determines both permeation and gating of the plant anion channel GCAC1.     

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.     

Anion-cation interactions in the pore of neuronal background chloride channels

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.     

Characterization of an anion channel in pituitary cells of Gillichthys mirabilis

Patch-clamp studies on pituitary cells from Gillichthys mirabilis show the presence of anion channels with selectivity CI > F = Br = I. These channels are voltage sensitive over the physiological range of membrane potentials and have a unitary conductance of 94 ± 15 pS in symmetrical KCl. Halides other than Cl on the cytoplasmic face of the membrane cause an increase in open probability (P(o)). DPC causes a dose dependent decrease in P(o) without affecting conductance. Sodium on the cytoplasmic face of the membrane causes a decrease in outward current.     

Nonselective ionic channels inAplysia neurones

Summary Single-channel recordings from outside-out patches ofAplysia neurones in K-free solutions revealed the presence in most membrane patches of ionic channels showing surprising selectivity properties, as deduced from reversal potential measurements. After complete substitution of external NaCl by mannitol (in the presence of internal CsCl), these channels are more permeable to Cl than to Cs, but are also slightly permeable to Cs:P _Cl/P _Cs=4. Furthermore, in the presence of external NaCl, their ability to discriminate cations from anions seems lower than in external mannitol. Substitutions of external Cl by various anions showed that the channels are more permeable to NO₃ than to Cl, and that they are appreciably permeable to isethionate, SO₄ and methanesulfonate. Their elementary conductance is about 100 pS in 600mm symmetrical Cl. However, different conductance states (usually 2 or 3) can often be detected in the same membrane patch. By using voltage ramps, we established theI–V curves corresponding to each of these states and found small but significant differences between the reversal potentials of each state.     

Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles

The electrical properties of the tonoplast of mature sugarbeet root vacuoles have been studied using the patch-clamp technique. In whole-vacuole recordings, the addition of 5 mM Mg-ATP to the external solution activated a proton-translocating ATPase which produced inward currents of up to 65 pA. Furthermore, we identified a voltage-dependent membrane conductance which activated at hyperpolarized (inside-negative) potentials and decreased at positive potentials. Outside-out membrane patches predominantly contained a channel which showed an increasing probability of opening at potentials more negative than about –20 mV. These channels can account for the macroscopic currents recorded in whole vacuoles. The permeability sequence of the channel for cations and anions was: P_K^staggered+ = P_Na⁺ >P_Ac⁻ >P_NO3⁻ >P_Mal²⁻ >P_Cl⁻. The unit conductance of this channel was about 70 pS in symmetrical 50 mM KCl and 180 pS in symmetrical 200 mM KCl solutions. Another channel type of smaller conductance (15 pS in 50 mM KCl) was also present, but its properties have not yet been studied. The permeability sequence of the nonselective channel corresponds to that found by tracer measurements in vacuole suspensions, implying that the channel studied may present the molecular pathway for the movement of ions across the tonoplast.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

The Effects of Alkali Metal Cations and Common Anions on the Frog Skin Potential

The effects on the potential difference across isolated frog skin (R. catesbeiana, R. pipiens) of changing the ionic composition of the bathing solutions have been examined. Estimates of mean values and precision are presented for the potential changes produced by substituting other alkali metal cations for Na at the outside border and for K at the inside border. In terms of ability to mimic Na at the outside border of bullfrog skin, the selectivity order is Li > Rb, K, Cs; at the outside border of leopard frog skin, Li > Cs, K, Rb. In terms of ability to mimic K at the inside border of bullfrog and leopard frog skin: Rb > Cs > Li > Na. Orders of anion selectivity in terms of sensitivity of the potential for the outside border of bullfrog skin are Br > Cl > NO₃ > I > SO₄, isethionate and of leopard frog skin are Br, Cl > I, NO₃, SO₄. An effect of the solution composition (ionic strength?) on the apparent Na-K selectivity of the outside border is described. The results of the investigation have been interpreted and discussed in terms of the application of the constant field equation to the Koefoed-Johnsen-Ussing frog skin model. These observations may be useful in constructing and testing models of biological ionic selectivity.     

Single anion channels reconstituted from cardiac mitoplasts

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. The appearance of anion rather than cation channels could be promoted by exposing the bilayers to osmotic gradients formed by Cl⁻ salts of large, relatively imperment, cations at a pH of 8.8. Two distinct activities were identified. These comprised a multisubstate anion channel of intermediate conductance (∼60 pS in 300vs. 50mm choline Cl, ∼100 pS in symmetric 150mm KCl), and a lower-conductance anion channel (∼25 or ∼50 pS in similar conditions), which only displayed two well-defined substates, at ∼25 and ∼50% of the fully open state. The larger channels were not simple multiples of the lower-conductance channels, but both discriminated poorly, and to a similar extent, between anions and cations (P_Cl ⁻/P_choline ⁺ ∼12, P_Cl ⁻/P_K ⁺∼8). The lower-conductance channel was only minimally selective between different anions (P_{NO 3} ⁻ (1.0)=P_Cl ⁻>P_Br ⁻>P_I ⁻>P_SCN ⁻(0.8)), and its conductance failed to saturate even in high (>1.0 M) activities of KCl. The channels were not obviously voltage dependent, and they were unaffected by 0.5 mM SITS, H₂O₂, propranolol, quinine or amitriptyline, or by 2 mM ATP, or by variations in pH (5.5–8.8). Ca²⁺ and Mg²⁺ did not alter single channel activity, but did modify single current amplitudes in the lower-conductance channel. This effect, together with voltage-dependent substate behavior, is described in the following paper.     

Mouse bestrophin-2 is a bona fide Cl(-) channel: identification of a residue important in anion binding and conduction

Bestrophins have recently been proposed to comprise a new family of Cl(-) channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl(-) channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca(2+)-activated Cl(-) current in all three cell lines (EC(50) for Ca(2+) = 230 nM). The permeability sequence was SCN(-): I(-): Br(-): Cl(-): F(-) (8.2: 1.9: 1.4: 1: 0.5). Although SCN(-) was highly permeant, its conductance was approximately 10% that of Cl(-) and SCN(-) blocked Cl(-) conductance (IC(50) = 12 mM). Therefore, SCN(-) entered the pore more easily than Cl(-), but bound more tightly than Cl(-). Mutations in S79 altered the relative permeability and conductance for SCN(-) as expected if S79 contributed to an anion binding site in the channel. P(SCN)/P(Cl) = 8.2 +/- 1.3 for wild-type and 3.9 +/- 0.4 for S79C. G(SCN)/G(Cl) = 0.14 +/- 0.03 for wild-type and 0.94 +/- 0.04 for S79C. In the S79 mutants, SCN(-) did not block Cl(-) conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN(-). Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET(+) or MTSES(-) increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl(-) conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.     

Anion and cation permeability of a chloride channel in rat hippocampal neurons

The ionic permeability of a voltage-dependent Cl channel of rat hippocampal neurons was studied with the patch-clamp method. The unitary conductance of this channel was approximately 30 pS in symmetrical 150 mM NaCl saline. Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz voltage equation indicate a Cl:Na permeability ratio of approximately 5:1 for conditions where there is a salt gradient. Many anions are permeant; permeability generally follows a lyotropic sequence. Permeant cations include Li, Na, K, and Cs. The unitary conductance does not saturate for NaCl concentrations up to 1 M. No Na current is observed when the anion Cl is replaced by the impermeant anion SO4. Unitary conductance depends on the cation species present. The channel is reversibly blocked by extracellular Zn or 9-anthracene carboxylic acid. Physiological concentrations of Ca or Mg do not affect the Na:Cl permeability ratio. The permeability properties of the channel are consistent with a permeation mechanism that involves an activated complex of an anionic site, an extrinsic cation, and an extrinsic anion.     

A synthetic prostone activates apical chloride channels in A6 epithelial cells

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br > NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.