CFTR in K562 human leukemic cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.     

The fast gating mechanism in ClC-0 channels

We investigate and then modify the hypothesis that a glutamate side chain acts as the fast gate in ClC-0 channels. We first create a putative open-state configuration of the prokaryotic ClC Cl- channel using its crystallographic structure as a basis. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to ClC-0 by replacing all the nonconserved polar and charged residues. Using this open-state channel model, we carry out molecular dynamics simulations to study how the glutamate side chain can move between open and closed configurations. When the side chain extends toward the extracellular end of the channel, it presents an electrostatic barrier to Cl- conduction. However, external Cl- ions can push the side chain into a more central position where, pressed against the channel wall, it does not impede the motion of Cl- ions. Additionally, a proton from a low-pH external solution can neutralize the extended glutamate side chain, which also removes the barrier to conduction. Finally, we use Brownian dynamics simulations to demonstrate the influence of membrane potential and external Cl- concentration on channel open probability.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

A GTP-binding protein activates chloride channels in a renal epithelium

Although G proteins have been shown to regulate cation channels, regulation of Cl- channels by G proteins has not been demonstrated directly. Accordingly, the objective of this study was to examine whether a G protein regulates Cl- channels in the apical membrane of rabbit kidney CCD cells grown in culture. Previous studies showed that this channel is activated by adenosine and protein kinase C and has a single channel conductance of 305 picosiemens. The PCl-:PNa+ is 9:1 and the PCl-:PHCO3- is 2:1 (Schwiebert, E.M., Light, D.B., Dietl, P., Fejes-Toth, G., Naray-Fejes-Toth, A., and Stanton, B. (1990) Kidney Int. 37,216). In the present study, Cl- channels in the apical membrane of CCD cells were studied by the patch clamp technique. GTP and guanosine 5'-O(3-thiophosphate) (GTP gamma S), a nonhydrolyzable analog of GTP, increased the single channel open probability (Po). In contrast, guanosine 5'-O-(2-thiophosphate), a nonhydrolyzable analog of GDP, and pertussis toxin (PTX) decreased the Po. GTP gamma S, but not GTP, reversed PTX inhibition of the channel. The alpha i-3-subunit of Gi increased the Po in both untreated and PTX-treated membrane patches. Because GTP gamma S activated the Cl- channel in the presence of H8, a protein kinase inhibitor, we conclude that the G protein does not activate the channel by stimulating a protein kinase. Thus, a PTX-sensitive G protein activates a Cl- channel in the apical membrane of renal CCD cells.     

G protein modulates thyroid hormone-induced Na(+) channel activation in ventricular myocytes

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T(3)) on Na(+) channel current (I(Na)) properties, I(Na) was recorded in adult guinea pig ventricular myocytes. T(3) (1 nM) acutely increased whole cell I(Na) and shifted the steady-state I(Na) inactivation curve dose dependently. When the pipette solution contained 100 microM GTP or GTPgammaS, the effect of T(3) on the whole cell I(Na) was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T(3) increased the open probability of single I(Na) by reducing the null probability. In the inside-out patch, T(3) effect was 10 times faster than that in whole cell and cell-attached patches while GTPgammaS was present and could be completely washed out. T(3) alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k(CO)) and reducing the null probability. GTPgammaS exposure only increased the number of functional channels. T(3) and GTPgammaS synergistically enhanced the channel open probability 5.8 +/- 0.5-fold by increasing k(CO), decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T(3) acts on the cytosolic side of the membrane and acutely activates I(Na). Pertussis toxin-sensitive G protein modulation greatly magnifies the T(3) effects on the channel kinetics and null probability, thereby increasing the channel open probability.     

Group IIIA-metal hydroxides indirectly neutralize the voltage sensor of the voltage-dependent mitochondrial channel, VDAC, by interacting with a dynamic binding site.

The voltage-dependent, anion-selective mitochondrial channel, VDAC, undergoes two different conformational changes from the open to a closed state under positive and negative applied electric fields. Micromolar quantities of aluminum hydroxide and other metal trihydroxides have recently been shown to be able to inhibit this voltage-dependent closure (Dill et al. (1987) J. Membr. Biol. 99, 187-196; Zhang and Colombini (1989) Biochim. Biophys. Acta 991, 68-78). It was suggested that the inhibition results from the neutralization of the positively charged voltage sensors by the metal species. In the present study, the dynamics of the metal-binding site accompanying channel closure was investigated by adding In(OH)3 to only one side of the membrane and examining its effect on the channel's gating processes. Indium added to open channels inhibited channel closure only when the metal-containing side was on the lower potential side of the applied field. If indium was added only to the higher-potential side, the channels closed and tended to remain closed after the field was abolished. The addition of metal hydroxide after closing the channels with a negative potential on the metal side did not result in channel re-opening as would be expected for sensor neutralization. Inhibition occurred immediately, however, if the channels were first allowed to open briefly. The closed-state selectivity seemed to be very similar in the absence or presence of the metal, indicating that the metal-binding sites are not located within the pore of the channel in the closed conformation. The results are consistent with a voltage-dependent translocation across the membrane of each of two metal-binding sites on VDAC. This translocation is tightly coupled with channel opening and closing.     

Antibody interaction with the amphotericin channel

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.     

A voltage-gated anion channel from the electric organ of Torpedo californica.

Addition of membrane vesicles prepared from the electric organ of Torpedo californica to the aqueous phase of a planar phospholipid bilayer system results in a large (up to 3 orders of magnitude) stepwise increase in membrane conductance. This increased conductance consists of two components: an ohmic background "leak" and a voltage-dependent, ideally anion-selective conductance. The anion conductance is low at voltages greater than +10 mV, rises sharply as the voltage becomes negative, and then saturates as the voltage becomes highly negative. (The trans side of the bilayer, to which vesicles are not added, is defined as ground.) Under high amplification, the anion conductance shows single channel behavior with a voltage-independent, single channel conductance of 13.9 +/- 0.1 pmho in 0.1 M Cl-. Furthermore, the anion channel, but not the background conductance, is inhibited by submillimolar concentrations of SITS and DIDS, two well known anion transport inhibitors. The inhibition is seen only when SITS or DIDS is added to the cis side. No cholinergic agents tested have any effect on the channel.     

Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6- trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP- ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.     

Small conductance chloride channels in the apical membrane of thyroid cells.

A small conductance chloride channel has been identified on the apical membrane of porcine thyroid cells using the patch-clamp technique. In cell attached membrane patches with NaCl in the pipette, the single channel conductance is 5.5 pS. The channel is highly selective for chloride over gluconate and iodide, and is impermeable to Na+, K+ and tetraethylammonium ions. The open state probability of the channel is not affected by voltage. The channel activity disappears after excision of the patch. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) did not affect the activity of the thyroid Cl- channels. Treatment of thyroid cells with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-chloro-cAMP) (0.5 mM) prior to giga-seal formation increased Cl- channel activity in the apical membrane of thyroid cells.     

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10- fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.     

Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.     

Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS

Mechanosensitive channels are a class of ubiquitous membrane proteins gated by mechanical strain in the cellular membrane. MscS, the mechanosensitive channel of small conductance, is found in the inner membrane of Escherichia coli and its crystallographic structure in an open form has been recently solved. By means of molecular dynamics simulations we studied the stability of the channel conformation suggested by crystallography in a fully solvated lipid (POPC) bilayer, the combined system encompassing 224,340 atoms. When restraining the backbone of the protein, the channel remained in the open form and the simulation revealed intermittent permeation of water molecules through the channel. Abolishing the restraints under constant pressure conditions led to spontaneous closure of the transmembrane channel, whereas abolishing the restraints when surface tension (20 dyn/cm) was applied led to channel widening. The large balloon-shaped cytoplasmic domain of MscS exhibited spontaneous diffusion of ions through its side openings. Interaction between the transmembrane domain and the cytoplasmic domain of MscS was observed and involved formation of salt bridges between residues Asp62 and Arg128; this interaction may be essential for the gating of MscS. K+ and Cl- ions showed distinctively different distributions in and around the channel.     

Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.     

G-proteins mediate intestinal chloride channel activation.

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain

Summary C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from thecis side (the side to which protein has been added) and only open channels from thetrans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.     

A calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells.

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.     

Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport

The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.     

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.