Reconstitution and regulation of an epithelial chloride channel

We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M_r 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (P_Cl/P_Na = 20.2; P_Na/P_K = 1) whose unit conductance is 62.4 ± 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01–1.0 M). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is intracellular, (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.     

Kinetic analysis of channel gating. Application to the cholinergic receptor channel and the chloride channel from Torpedo californica.

Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.     

CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.     

Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function

It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K⁺ channels to systematically examine the impact of bilayer composition on the pore module of single K⁺ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K⁺ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.     

Burst properties of a supergated double-barrelled chloride ion channel

The chloride selective channel from Torpedo electroplax, ClC-0, is the prototype of a large gene family of chloride channels that behave as functional dimers, with channel currents exhibiting two non-zero conductance levels. Each pore has the same conductance and is controlled by a subgate, and these have seemingly identical fast gating kinetics. However, in addition to the two subgates there is a single slower 'supergate' which simultaneously affects both channels. In the present paper, we consider a six state Markov model that is compatible with these observations and develop approximations as well as exact results for relevant properties of groupings of openings, known as bursts. Calculations with kinetic parameter values typical of ClC-0 suggest that even simple approximations can be quite accurate. Small deviations from the assumption of independence within the model lead to marked changes in certain predicted burst properties. This suggests that analysis of these properties may be helpful in assessing independence/non-independence of gating in this type of channel. Based on simulations of models of both independent and non-independent gating, tests using binomial distributions can lead to false conclusions in each situation. This is made more problematic by the difficulty of selecting an appropriate critical time in defining a burst empirically.     

Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart

Mitochondrial ATP-regulated potassium (mitoKATP) channels play an important role in cardioprotection. Single channel activity was measured after reconstitution of inner mitochondrial membranes from bovine myocardium into a planar lipid bilayer. After incorporation, the potassium channel was recorded with a mean conductance of 103+/-9 pS. The channel activity was inhibited by ATP/Mg and activated by GDP. Magnesium ions alone affected, in a dose dependent manner, both the channel conductance and the open probability. Magnesium ions regulated the mitoKATP channel only when added to the trans compartment. We conclude that Mg2+ regulates the cardiac mitoKATP channel from the matrix site by affecting both the channel conductance and gating.     

Thermodynamic and kinetic studies of the gating behavior of a K+- selective channel from the sarcoplasmic reticulum membrane

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.     

Syringomycin E channel: a lipidic pore stabilized by lipopeptide?

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.     

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.     

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.     

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.     

Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis

Oocytes of the South African clawed toad Xenopus laevis possess in their plasma membrane a so-called stretch-activated cation channel (SAC) which is activated by gently applying positive or negative pressure (stretch) to the membrane patch containing the channels. We show here that this mechanosensitive channel acted as a spontaneously opening, stretch-independent non-selective cation channel (NSCC) in more than half of the oocytes that we investigated. In 55% of cell-attached patches (total number of patches, 58) on 30 oocytes from several different donors, we found NSCC opening events. These currents were increased by elevating the membrane voltage or raising the temperature. NSCC and SAC currents shared some properties regarding the relative conductances of Na+>Li+>Ca2+, gating behaviour and amiloride sensitivity. Stretch-independent currents could be clearly distinguished from stretch induced SAC currents by their voltage and temperature dependence. Open events of NSCC increased strongly when temperature was raised from 21 to 27 degrees C. NSCC currents could be partly inhibited by high concentrations of extracellular Gd3+ and amiloride (100 and 500 microM, respectively). We further show exemplarily that NSCC can seriously hamper investigations when oocytes are used for the expression of foreign ion channels. In particular, NSCC complicated investigations on cation channels with small conductance as we demonstrate for a 4 pS epithelial Na+ channel (ENaC) from guinea pig distal colon. Our studies on NSCCs suggest the involvement of these channels in oocyte temperature response and ion transport regulation. From our results we suggest that NSCC and SAC currents are carried by one protein operating in different modes.     

An inwardly rectifying chloride channel in ragweed-sensitized canine tracheal epithelial cells

The single channel inside-out patch clamp technique was used to characterize ion channels in the apical membranes of ragweed-sensitized and control canine tracheal epithelial cells maintained in primary culture. Patches were obtained from single isolated cells or from cells at the edges of confluent sheets. A new type of chloride channel was seen in sensitized cells but not in control cells. The channel showed inward rectification in symmetric chloride solutions with conductance varying from 95 pS to 52 pS over the range of –60 mV to 60 mV membrane potential. Channel gating was voltage dependent with maximal opening at about –30 mV Kinetic analysis showed that distributions of closed and open times could both be well fitted by the sums of three exponential components. Rate constants for transitions between the states of a linear kinetic model were calculated, with only one rate being significantly voltage dependent. The possible significance of this channel is discussed. Offprint requests to: A. S. French     

MscS, the bacterial mechanosensitive channel of small conductance

The mechanosensitive channel of small conductance, MscS, is one of the most extensively studied MS channels to date. Past and present research involves the discovery of its physiological role as an emergency valve in prokaryotes up to detailed investigations of its conductive properties and gating mechanism. In this review, we summarize the findings on its structure and function obtained by experimental and theoretical approaches. A special focus is given to its pharmacology, since various compounds have been shown to affect the activity of this channel. These compounds have particularly been helpful for understanding the interaction of MscS with the lipid bilayer, as well as recognizing the potential of this channel as a target for novel types of antibiotics.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Voltage-dependent gating properties of the channel formed byE. coli hemolysin in planar lipid membranes

Escherichia coli hemolysin forms cation selective, ion-permeable channels of large conductance in planar phospholipid bilayer membranes. The pore formation mechanism is voltage dependent resembling that of some colicins and of diphtheria toxin: pores open when negative voltages are applied and close with positive potentials. The pH dependence of this gating process suggests that it is mediated by a negative fixed charge present in the lumen of the pore. A simple physical model of how the channel opens and closes in response to the applied voltage is given.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

Purified ryanodine receptor from rabbit skeletal muscle is the calcium- release channel of sarcoplasmic reticulum

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.