Single chloride channels from Torpedo electroplax. Activation by protons

Single-channel fluctuations of a chloride-specific channel from Torpedo californica electroplax were studied with high current and time resolution. Channels were incorporated into virtually solvent-free planar bilayer membranes formed from phospholipid monolayers, and the substructure of the open channel was analyzed. The single channel displays three well-defined substates of conductances 0, 10, and 20 pS in 200 mM Cl-. These three substates are interpreted in terms of a dimeric channel complex composed of two identical "protochannels" gating independently in parallel on a time scale of milliseconds, but coupled together by a bursting process on a time scale of seconds. The probability of forming an open protochannel is voltage dependent and is increased strongly as aqueous pH is lowered. Variations of pH are effective only on the same side of the bilayer as the addition of electroplax vesicles. The dependence of single-channel kinetics on pH and voltage lead to a minimal four-state model in which both open and closed states can be protonated on a residue that changes its pK from 6 to 9 upon opening of the protochannel.     

L-glutamate activates excitatory and inhibitory channels in Drosophila larval muscle

Muscle fibers from Drosophila larvae show an L-glutamate-sensitive membrane potential. Bath-applied L-glutamate depolarizes the muscle in the range from 0.5 to 20 microM. Greater concentrations of the agonist repolarize the fibers. The repolarizing effect disappears if chloride is replaced by sulfate in the external medium. Intracellular recordings show the occurrence of depolarizing and hyperpolarizing spontaneous miniature postsynaptic potentials (smpp). Patch-clamp studies indicate the presence of two types of receptor channels: (i) an anion-selective channel activated by both L-glutamate and GABA. In outside out-patches, bathed in symmetrical 140 mM Cl- and 200 microM GABA, the channel displays conductance substates of 40, 80 and 110 pS. In the presence of 200 microM L-glutamate only the 40 and 80 pS substates are observed; (ii) a cation-selective channel activated only by L-glutamate that has a conductance of 104 pS in cell-attached patches (128 mM Na+ outside). The presence of these two types of receptor channels in Drosophila muscle may explain the effect of bath-applied L-glutamate on membrane potential and the presence of inhibitory and excitatory smpp.     

ATP inhibition and rectification of a Ca2+-activated anion channel in sarcoplasmic reticulum of skeletal muscle.

We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl-) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca2+ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (approximately 1 mM) inhibited only when Cl- flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K(I) of 0.4-1 mM at -40 mV (Hill coefficient approximately 2), which increased at more negative potentials. Luminal ATP inhibited with a K(I) of approximately 2 mM at +40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (approximately 1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl- from cytoplasm to SR lumen in vivo, and balance electrogenic Ca2+ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.     

The sodium channel activator Brevetoxin-3 uncovers a multiplicity of different open states of the cardiac sodium channel.

The interaction of Brevetoxin 3 (Pbtx-3), a sodium channel activator, with the cardiac sodium channel was studied at the single channel level. It was found that Pbtx-3 (20 microM) shifted steady-state activation to negative potentials, without major effects on the time course of macroscopic activation or macroscopic currents decay, as calculated from averaged single-channel records. Single-channel open times were found to be prolonged. Under the influence of the toxin, sodium channel openings could be observed frequently even at maintained depolarisation. These openings occurred to at least nine different subconductance levels of the open state with smaller conductivities than the maximal one and differed in their open times. Current amplitudes of these open substates were found to cluster around certain amplitude values. Appearance of substates at maintained depolarisation was dependent on the transmembrane potential (Em): Substates with smaller conductivity appeared more frequently at lower Em values whereas at higher Em values substates with higher conductivity values dominated. Furthermore, it was demonstrated that appearance of substates did not result from incomplete recovery from inactivation. From these observations it was concluded that the open substates observed correspond to different conformational states of the channel's activation gates. Under physiological conditions, when the sodium channel opens directly from its closed state these 'incomplete'-open states of the cardiac sodium channel are obscured by fast gating transitions between the corresponding, electrically silent, preopen states. Thus, Pbtx-3 acts mainly via stabilisation of the channel's preopen and different open states. A classification of sodium channel modifiers, based on their interaction with different conformational states of the channel is suggested.     

The cardiac sodium channel shows a regular substate pattern indicating synchronized activity of several ion pathways instead of one

Cardiac sodium channel substates were induced by using different gating modifiers, namely S-DPI 201-106 (s), toxin II from Anemonia sulcata (a), veratridine (v) and mixtures of these agents (s + v, a + v). Current ratios (normalized substate currents), slope conductances, reversal potentials and saturation characteristics were evaluated for the individual channel substates. The results can be summarized as follows: (i) Current ratios fell into a pattern of six equidistant values (I to VI) irrespective of the modification applied (0.20, 0.34, 0.51, 0.69, 0.85, 1.00). Slope conductances, determinable for substates II, V and VI (4.8, 11.7 and 14.0, respectively), are also consistent with six conductance substates which are integer multiples of a smallest conductance (state I). (ii) The permeability ratio PNa+/PK+ (i.e., reversal potential of substate currents) of the sodium channel was conserved both for different modifications, i.e., by s, a, s + v and a + v, and for the different substates (at least for II, IV and VI) observed for each modification. (iii) Sodium binding to the channel is substate independent. Analysis of slope conductances of states II and VI for three sodium chloride concentrations (71.5, 140 and 303 mM) revealed different maximal conductances (geVImax = 2.9.geIImax) but similar apparent affinities for sodium (KNa + VI = 286 mM; KNa + II = 303 mM). These findings are shown to seriously challenge the commonly unquestioned conception that 'single-current events' reflect ion passage through only one single pathway. The alternative view, that not one pore, but either six or three pores with synchronized gating ('oligochannel') underlie 'single-channel events', is shown to readily account for the observed substate properties and appears not to contradict known properties of 'the sodium channel'. This fundamentally new view of the sodium channel aims to invoke further efforts to distinguish between conceptually distinct models of structure-function relationships for a variety of channels which show multiple substates and conserved ion selectivity.     

Reconstitution of ionic channels from inner and outer membranes of mammalian cardiac nuclei.

Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.     

Hofmeister effect in ion transport: reversible binding of halide anions to the roflamycoin channel.

We have studied the anion-dependent gating of roflamycoin ion channels using spectral analysis of noise in currents through multichannel planar lipid bilayers. We have found that in addition to low frequency current fluctuations that may be attributed to channel switching between open and closed conformations, roflamycoin channels exhibit a pronounced higher frequency noise indicating that the open channel conductance has substates with short lifetimes. This noise is well described by a Lorentzian spectrum component with a characteristic cutoff frequency that depends on the type of halide anions according to their position in the Hofmeister series. It is suggested that transitions between the substates correspond to a reversible ionization of the channel by a penetrating anion that binds to the channel structure, more chaotropic anions being bound for longer times. Within a framework of a two-substate model, the duration of the substate with reduced electrostatic barrier for cation current varies exponentially with anion electron polarizability. This explains two features of the roflamycoin channel reported earlier: the increase in apparent single-channel conductance along the series F- < Cl- < Br- < I- and the reverse of channel selectivity from anionic for KF to cationic for KI.     

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.     

Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A comparison is made of two types of chloride-selective channel in skeletal muscle sarcoplasmic reticulum (SR) vesicles incorporated into lipid bilayers. The I/V relationships of both channels, in 250/50 mM Cl- (cis/trans), were linear between -20 and +60 mV (cis potential,) reversed near Ecl and had slope conductances of approximately 250 pS for the big chloride (BCl) channel and approximately 70 pS for the novel, small chloride (SCl) channel. The protein composition of vesicles indicated that both channels originated from longitudinal SR and terminal cisternae. BCl and SCl channels responded differently to cis SO4(2-) (30-70 mM), 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (8-80 microM) and to bilayer potential. The BCl channel open probability was high at all potentials, whereas SCl channels exhibited time-dependent activation and inactivation at negative potentials and deactivation at positive potentials. The duration and frequency of SCl channel openings were minimal at positive potentials and maximal at -40 mV, and were stationary during periods of activity. A substate analysis was performed using the Hidden Markov Model (S. H. Chung, J. B. Moore, L. Xia, L. S. Premkumar, and P. W. Gage, 1990, Phil. Trans. R. Soc. Lond. B., 329:265-285) and the algorithm EVPROC (evaluated here). SCl channels exhibited transitions between 5 and 7 conductance levels. BCl channels had 7-13 predominant levels plus many more short-lived substates. SCl channels have not been described in previous reports of Cl- channels in skeletal muscle SR.     

短杆菌肽A-DMPC通道内离子输运的分子动力学模拟

用最近提出的构建膜体系初始构象的有效方法 ,构建了在DMPC脂膜环境下短杆菌肽A通道模型 (GA -DMPC)。通过对Na 、Ca2 、Cl-三种不同离子在GA -DMPC通道内不同位置的分子动力学模拟 ,研究离子在通道内输运过程中与通道及通道内水分子的相互作用 ,从分子动力学的角度阐明离子在通道内的输运机制。主要计算结果表明 :(1)离子在通道内的输运使GA的构象发生变化 ,GA的柔性是离子在通道内通透的重要因素 ;(2)Cl- 离子可扩大通道半径 ,Na 离子和Ca2 离子则减小通道半径。Cl-离子不能在GA通道内通透 ;(3)离子的出现使通道内水分子的偶极方向发生变化。上述结果均与实验相符。     

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.     

A large conductance ion channel in the nuclear envelope of a higher plant cell.

To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of +/- 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.     

Multichannel recordings from membranes which contain gap junctions. II. Substates and conductance shifts.

Substates which can last up to several seconds are found in the 100-pS channel of the earthworm septum, a putative gap junction channel. The conductance of these substates is highly variable from preparation to preparation, and they are found at almost every fraction of the whole channel conductance. Another phenomenon seen in multichannel recordings is the "conductance shift": here the current passed by several open channels differs from an integral multiple of the current when only one channel is open. These shifts can be modelled by 1) a resistance in series with the channel or 2) long-lived substates. Each of these models fails in particular cases to explain either the magnitude or direction of the shifts. It is possible that both effects are simultaneously present.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.     

Subconductance states of Cx30 gap junction channels: data from transfected HeLa cells versus data from a mathematical model

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, gamma(j,substate), were unevenly distributed between the main-state and the residual-state conductance (gamma(j,main state) = 141 pS, gamma(j,residual state) = 21 pS). Activation of the first subgate reduced the channel conductance by approximately 30%, and activation of subsequent subgates resulted in conductance decrements of 10-15% each. Current transitions between the states were fast (<2 ms). Substate events were usually demarcated by transitions from and back to the main state; transitions among substates were rare. Hence, subgates are recruited simultaneously rather than sequentially. The incidence of substate events was larger at larger gradients of V(j). Frequency and duration of substate events increased with increasing number of synchronously activated subgates. Our mathematical model, which describes the operation of gap junction channels, was expanded to include channel substates. Based on the established V(j)-sensitivity of gamma(j,main state) and gamma(j,residual state), the simulation yielded unique functions gamma(j,substate) = f(V(j)) for each substate. Hence, the spacing of subconductance levels between the channel main state and residual state were uneven and characteristic for each V(j).     

Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK

The effects of various intracellular anions on the G protein (GK)-mediated activation of the muscarinic K+ (KACh) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5 microM) in the pipette, 1 microM GTP caused different magnitudes of KACh channel activation in internal solutions containing different anions. The order of potency of anions to induce the KACh channel activity at 0.5 microM ACh and 1 microM GTP was Cl- greater than or equal to Br- greater than 1-. In the SO4(2-) or aspartic acid internal solution, no channel openings were induced by 1 microM GTP with 0.5 microM ACh. In both the Cl- and SO4(2-) internal solutions (with 0.5 microM ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (Kd) was 0.2 microM in the Cl- and 10 microM in the SO4(2-) solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-o-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO4(2-) solutions; i.e., the Hill coefficient was approximately 3-4 and the Kd was approximately 0.06-0.08 microM in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO4(2-) solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of GK, resulting in higher sensitivity of the KACh channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (greater than 1 microM) or ATP (greater than 1 mM) alone caused activation of the KACh channel, while neither occurred in the SO4(2-) solution. These observations suggest that the activation of the KACh channel by the basal turn-on reaction of GK or by phosphate transfer to GK by nucleoside diphosphate-kinase may depend at least partly on the intracellular concentration of Cl-.     

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.     

An outer membrane protein undergoes enthalpy- and entropy-driven transitions

β-Barrel membrane proteins often fluctuate among various open substates, yet the nature of these transitions is not fully understood. Using temperature-dependent, single-molecule electrophysiology analysis, along with rational protein design, we show that OccK1, a member of the outer membrane carboxylate channel from Pseudomonas aeruginosa, features a discrete gating dynamics comprising both enthalpy-driven and entropy-driven current transitions. OccK1 was chosen for the analysis of these transitions, because it is a monomeric transmembrane β-barrel of a known high-resolution crystal structure and displays three distinguishable, time-resolvable open substates. Native and loop-deletion OccK1 proteins showed substantial changes in the activation enthalpies and entropies of the channel transitions, but modest alterations in the equilibrium free energies, confirming that the system never departs from equilibrium. Moreover, some current fluctuations of OccK1 indicated a counterintuitive, negative activation enthalpy, which was compensated by a significant decrease in the activation entropy. Temperature scanning of the single-channel properties of OccK1 exhibited a thermally induced switch of the energetically most favorable open substate at the lowest examined temperature of 4 °C. Therefore, such a semiquantitative assessment of the current fluctuation dynamics not only demonstrates the complexity of channel gating but also reveals distinct functional traits of a β-barrel outer membrane protein under different temperature circumstances.     

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.     

Phosphorylation-activated chloride channels in human skin fibroblasts

A chloride-selective channel has been found using patch-clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl- channel found in airway epithelia. As in the case of epithelial Cl- channels, Cl- channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E2 evoke Cl- channel activity in cell-attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg2+ to the internal membrane surface of excised, inside-out patches. The Cl- channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl- channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.