Anion channels activated by adrenaline in cardiac myocytes

In heart cells, the catecholamine-activated cyclic AMP system regulates calcium and potassium channels. We report here a novel class of chloride channels that can be activated by adrenaline in mammalian ventricular cells. Like the agonist-activated Cl- channel currents of airway and colonic epithelial cells, the cardiac Cl(-)-channel current shows outward rectification. But the unit conductance of cardiac Cl- channels is smaller than that of epithelial Cl- channels. The cardiac Cl- channel is functionally voltage-independent, in contrast to the Cl- channel in colonic epithelial cells. This channel could be responsible for the beta-catecholamine-induced increase in cardiac membrane conductance that has been attributed to activation of a Cl- current. Thus, sympathetic control of cardiac electrical activity involves not only the voltage-dependent, excitation-related cation channels, but also anion channels that generate a steady current.     

A chloride channel widely expressed in epithelial and non-epithelial cells.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.     

Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels

ClC Cl- channels make up a large molecular family, ubiquitous with respect to both organisms and cell types. In eukaryotes, these channels fulfill numerous biological roles requiring gated anion conductance, from regulating skeletal muscle excitability to facilitating endosomal acidification by (H+)ATPases. In prokaryotes, ClC functions are unknown except in Escherichia coli, where the ClC-ec1 protein promotes H+ extrusion activated in the extreme acid-resistance response common to enteric bacteria. Recently, the high-resolution structure of ClC-ec1 was solved by X-ray crystallography. This primal prokaryotic ClC structure has productively guided understanding of gating and anion permeation in the extensively studied eukaryotic ClC channels. We now show that this bacterial homologue is not an ion channel, but rather a H+-Cl- exchange transporter. As the same molecular architecture can support two fundamentally different transport mechanisms, it seems that the structural boundary separating channels and transporters is not as clear cut as generally thought.     

Inactivation of muscle chloride channel by transposon insertion in myotonic mice.

MYOTONIA (stiffness and impaired relaxation of skeletal muscle) is a symptom of several diseases caused by repetitive firing of action potentials in muscle membranes. Purely myotonic human diseases are dominant myotonia congenita (Thomsen) and recessive generalized myotonia (Becker), whereas myotonic dystrophy is a systemic disease. Muscle hyperexcitability was attributed to defects in sodium channels and/or to a decrease in chloride conductance (in Becker's myotonia and in genetic animal models). Experimental blockage of Cl- conductance (normally 70-85% of resting conductance in muscle) in fact elicits myotonia. ADR mice are a realistic animal model for recessive autosomal myotonia. In addition to Cl- conductance, many other parameters are changed in muscles of homozygous animals. We have now cloned the major mammalian skeletal muscle chloride channel (ClC-1). Here we report that in ADR mice a transposon of the ETn family has inserted into the corresponding gene, destroying its coding potential for several membrane-spanning domains. Together with the lack of recombination between the Clc-1 gene and the adr locus, this strongly suggests a lack of functional chloride channels as the primary cause of mouse myotonia.     

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.     

Control of dynamic CFTR selectivity by glutamate and ATP in epithelial cells

Cystic fibrosis is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. Phosphorylation and ATP hydrolysis are generally believed to be indispensable for activating CFTR. Here we report phosphorylation- and ATP-independent activation of CFTR by cytoplasmic glutamate that exclusively elicits Cl-, but not HCO3-, conductance in the human sweat duct. We also report that the anion selectivity of glutamate-activated CFTR is not intrinsically fixed, but can undergo a dynamic shift to conduct HCO3- by a process involving ATP hydrolysis. Duct cells from patients with DeltaF508 mutant CFTR showed no glutamate/ATP activated Cl- or HCO3- conductance. In contrast, duct cells from heterozygous patients with R117H/DeltaF508 mutant CFTR also lost most of the Cl- conductance, yet retained significant HCO3- conductance. Hence, not only does glutamate control neuronal ion channels, as is well known, but it can also regulate anion conductance and selectivity of CFTR in native epithelial cells. The loss of this uniquely regulated HCO3- conductance is most probably responsible for the more severe forms of cystic fibrosis pathology.     

Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.     

X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.

The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 A, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with alpha-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.     

Gating of a muscle K+ channel and its dependence on the permeating ion species

In excitable cells, ions permeate the cell membrane through ionic channels, some of which open and close in response to changes in the potential difference across the membrane. It has been supposed that this opening and closing (or gating) process is largely independent of the permeating ion. However, we show here that the gating of the resting potassium permeability of frog skeletal muscle depends on the species of ion which carries current across the membrane. The potassium permeability investigated allows K+ to move in across the membrane more easily than out. This property is known as inward or anomalous rectification and is shared by cell membranes of skeletal muscle, egg and certain other cells. In both egg cells and skeletal muscle fibres, the group IIIB metal ion Tl+, which can replace K+ in several other systems in experimental conditions, also permeates the inward rectifier. Indeed, Tl+ is more permeant than K+ (refs 8, 9). However, when Tl+ carries current inwards across the membrane, the inward rectifier inactivates over a brief period when the membrane is hyperpolarized, whereas when K+ carries current, the permeability increases with time under hyperpolarization.     

Single postsynaptic channel currents in tissue cultured muscle

Some of the most compelling evidence for the existence of ionic channels in cell membranes comes from direct recording of quantised current jumps generated by the opening and closing of individual channels. Single-channel jumps have been extensively studied for lipid bilayer membranes doped with various channel-forming additives. Recently agonist-induced single-channel currents were detected in denervated frog muscle by use of extracellular electrodes, which can isolate the current from a small area of membrane. The current jumps provide a means for the direct test of many of the inferences about ionic channels which have come from electrical noise analysis. In this report we present measurements of single-channel currents induced by the agonist carbamylcholine in tissue-cultured mammalian muscle. These measurements confirm the earlier noise studies on tissue culture preparations. Recordings of single-channel currents induced by the agonist, suberyldicholine, in avian muscle are presented by Nelson and Sachs.     

Facilitation of voltage-gated ion channels in frog neuroglia by nerve impulses

The functions of glial cells in the nervous system are not well defined, with the exception of myelin production by oligodendrocytes, uptake of amino-acid synaptic transmitters, and a contribution to extracellular potassium homeostasis. Neuroglia have receptors for neurotransmitters which may be involved in neuron-glia interactions. Recent studies have demonstrated voltage-gated ion channels in glial membranes. In a study of the optic nerve of the frog, small areas of the surface were examined with the loose patch-clamp method, and voltage-gated Na+ and K+ channels, presumably located in the membranes of the astrocytes forming the glia limitans, were identified. We now report that nerve impulses in the axons of the frog optic nerve transiently alter the properties of the voltage-dependent membrane channels of the surface glial cells (astrocytes), a demonstration of a new form of neuron-glia interaction.     

A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.     

Conductances of single ion channels opened by nicotinic agonists are indistinguishable

Hypotheses concerning the mechanism by which acetylcholine-like agonists cause ion channels to open often suppose that the receptor-ionophore complex can exist in either of two discrete conformations, open and shut. On the basis of noise analysis it has been reported that certain agonists open ion channels of lower conductance than usual, though many potent agonists give similar conductances, and hence that differences in the conductance of ion channels opened by different agonists may contribute to differences in efficacy. Here we have reinvestigated this question by recording single ion channel currents evoked by acetylcholine-like agonists on embryonic rat muscle in tissue culture and on adult frog muscle endplate. Ten different agonists (Fig. 1) were tested, including several that noise analysis has suggested have a low conductance. The single-channel conductance was found to be the same, within a few per cent, for all 10 agonists. It seems that noise analysis has given erroneously low conductances in some cases. Therefore efficacy differences do not depend on differences in single-channel conductance evoked by various agonists but presumably on the position of the open-shunt equilibrium of the agonist-channel complexes.     

Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes

The concept of purinergic neurotransmission, first proposed by Burnstock, has been confirmed in various cell types. We show here, by the patch-clamp method, that external ATP in micromolar concentrations (1-100 microM) activates cation channels in the membranes of fusion-competent myoblasts and myotubes. In cell-attached membrane patches of myoblasts and myotubes the mean number of simultaneously activated channels increases with time after external ATP application. In myoblasts only one population of channels having a mean single-channel conductance of gamma=43 pS was found, while in myotubes two populations with gamma 1=48 pS and gamma 2=20 pS were observed. Treatment of myotube membranes with acetylcholine (ACh) or carbachol resulted in two populations of channels which had conductance values and voltage-dependent mean channel lifetimes similar to those produced in response to ATP. The results show that embryonic skeletal muscle cells contain cation channels sensitive to ATP and provide evidence for a neurotransmitter-like action of ATP on these cells.     

Activation of the epithelial Na+ channel (ENaC) requires CFTR Cl- channel function

It is increasingly being recognized that cells coordinate the activity of separate ion channels that allow electrolytes into the cell. However, a perplexing problem in channel regulation has arisen in the fatal genetic disease cystic fibrosis, which results from the loss of a specific Cl- channel (the CFTR channel) in epithelial cell membranes. Although this defect clearly inhibits the absorption of Na+ in sweat glands, it is widely accepted that Na+ absorption is abnormally elevated in defective airways in cystic fibrosis. The only frequently cited explanation for this hypertransport is that the activity of an epithelial Na+ channel (ENaC) is inversely related to the activity of the CFTR Cl- channel. However, we report here that, in freshly isolated normal sweat ducts, ENaC activity is dependent on, and increases with, CFTR activity. Surprisingly, we also find that the primary defect in Cl- permeability in cystic fibrosis is accompanied secondarily by a Na+ conductance in this tissue that cannot be activated. Thus, reduced salt absorption in cystic fibrosis is due not only to poor Cl- conductance but also to poor Na+ conductance.     

The appearance of single-ion channels in unmodified lipid bilayer membranes at the phase transition temperature

Ion-specific channels in artificial membranes have been formed by the addition of gramicidin A, alamethicin, polyene antibiotics and some proteins to the solution surrounding the bilayer lipid membrane. Until now there have been no reports of single-ion channels in unmodified lipid membranes. We have now studied the electrical conductance of planar lipid bilayers membranes made of synthetic distearoylphosphorylcholine (DSPC). Current fluctuations of amplitude approximately 1pA and duration approximately 1 s have been discovered at phase transition temperature, which shows that the appearance of ionic channels may be the result of lipid domain interactions. This would explain the dramatic increase in ion permeability observed in liposomes during phase transition. We suggest that these channels could conduct the transmembrane ionic current in biological membranes without the involvement of peptides and proteins.     

Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons

Glycine is an important inhibitory transmitter in the brainstem and spinal cord. In the trigeminal subnucleus caudalis (medullary dorsal horn) and in the spinal dorsal horn (the relaying centres for processing pain and sensory information), glycine inhibits the glutamate-evoked depolarization and depresses firing of neurons. The binding of glycine to its receptor produces a large increase in Cl- conductance, which causes membrane hyperpolarization. The selectivity and gating properties of glycine receptor channels have been well characterized; the glycine receptor molecules have also been purified. The amino-acid sequence, deduced from complementary DNA clones encoding one of the peptides (the 48K subunit), shows significant homology with gamma-aminobutyric acid A (GABAA) and nicotinic acetylcholine receptor subunits, suggesting that glycine receptors may belong to a superfamily of chemically gated channel proteins. However, very little is known about the modulation of glycine receptor channels. We have investigated the regulation of strychnine-sensitive glycine receptor channels by cyclic AMP-dependent protein kinase in neurons isolated from spinal trigeminal nucleus of rat and report here that the protein kinase A dramatically increased the glycine-induced Cl- currents by increasing the probability of the channel openings. GS protein, which is sensitive to cholera toxin, was involved in the modulation.     

The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.     

Potassium channels in the nodal membrane of rat myelinated fibres

Following some preliminary reports, mammalian fibres from rabbit and rat have recently been successfully studied in detail by means of the voltage clamp. The early transient or sodium conductance system was found to be similar to that in frog and squid axons. However, the delayed conductance or potassium currents were found to be negligible. Only after chemical and osmotic manipulations, which were said to expose channels buried under the myelin, did Chiu and Ritchie find delayed currents in rabbit fibres. If confirmed, this would mean that the membrane conductance system of mammalian fibres is so different from that of invertebrate and amphibian axon models as to make the data base gathered from amphibian myelinated fibres (frog and toad) and invertebrate giant axons (squid and myxicola) irrelevant to human nd other mammalian fibres. However, we show here that it is possible to find in the normal nodal membrane of rat myelinated fibres potassium currents that flow through channels which are similar in many respects to those found in the frog node of squid axons.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.