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.     

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.     

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.     

Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

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.     

Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart

Acetylcholine (ACh) released on vagal stimulation reduces the heart rate by increasing K+ conductance of pacemaker cells in the sinoatrial (S-A) node. Fluctuation analysis of ACh-activated currents in pacemaker tissue showed this to be due to opening of a separate class of K+ channels gated by muscarinic ACh receptors (m-AChRs). On the other hand, it has been suggested that m-AChRs may simply regulate the current flow through inward rectifying resting K+ channels (gk1). We report here the measurement of ACh-activated single channel K+ currents and of resting K+ channel currents in isolated cells of the atrioventricular (A-V) and S-A node of rabbit heart. The results show that the ACh-dependent K+ conductance increase in nodal cells is mediated by K+ channels which are different in their gating and conductance properties from the inward rectifying resting K+ channels in atrial and ventricular cells. The resting K+ channels in nodal cells are, however, similar to those activated by ACh.     

A Cl- conductance activated by hyperpolarization in Aplysia neurones

Although many voltage-gated cation channels have been described and extensively studied in biological membranes, there are very few examples of voltage-gated anion channels. Chloride conductances activated by depolarization have been observed in skate electroplaque and in frog and chick skeletal muscle. A Cl- conductance activated by hyperpolarization has been suggested both for frog muscle treated with acid (pH 5) solutions, and for crayfish muscle where it could account for the fact that the pronounced inward-going rectification of the I-V curve disappears if the fibres have been soaked in a Cl(-)-free solution. More recently, voltage-dependent anion channels extracted from biological membranes have been incorporated into artificial membranes. I now report that in Aplysia neurones, and in particular those in which the internal Cl- concentration has been increased, a Cl- conductance can be observed which is slowly activated by hyperpolarization and shows a vary steep voltage dependence. This time- and voltage-dependent Cl- conductance probably exists also in many other cells. Its presence might explain why it is difficult when using KCl-filled microelectrodes to maintain prolonged hyperpolarizations. This Cl- conductance constitutes a new type of inward-going rectification distinct both from the classical "anomalous rectification' which involves selective K+ channels and from the current termed if in heart muscle that is presently attributed to a cationic conductance.     

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.     

Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.     

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.     

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.     

Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.     

Chloride and potassium channels in cystic fibrosis airway epithelia

Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.     

Beta-adrenoceptor agonists increase membrane K+ conductance in cardiac Purkinje fibres

Hormonal modulation of the ionic conductance of cell membranes is a topic of considerable current interest; it has a major role, for example, in the improved performance of the vertebrate heart elicited by sympathetic nerve stimulation or by circulating catecholamines, an effect involving enhanced calcium influx. beta-Agonist catecholamines also abbreviate the action potential of cardiac Purkinje fibres, and increase the resting potential in a variety of cells, including cardiac cells, a hyperpolarization usually attributed to stimulation of the electrogenic Na+/K+ pump. We show here that nanomolar concentrations of beta-catecholamines cause hyperpolarization of cardiac Purkinje fibres, not by increasing Na+/K+ pump current, but by increasing resting membrane K+ conductance. The hyperpolarization and shortening of the action potential should increase availability of Na+ channels and reduce the refractory period, effects tending to safeguard impulse propagation through the ventricular conducting system despite the increased heart rate caused by beta-catecholamine action on the sinus node pacemaker.     

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.     

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.     

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.     

Beta-adrenergic modulation of calcium channels in frog ventricular heart cells

Adrenergic modulation of calcium channels profoundly influences cardiac function, and has served as a prime example of neurohormonal regulation of voltage-gated ion channels. Channel modulation and increased Ca influx are mediated by elevation of intracellular cyclic AMP and protein phosphorylation. The molecular mechanism of the augmented membrane Ca conductance has attracted considerable interest. An increase in the density of functional channels has often been proposed, but there has previously been no direct evidence. Single-channel recordings show that isoprenaline or 8-bromocyclic AMP increase the proportion of time individual channels spend open by prolonging openings and shortening the closed periods between openings. To look for an additional contribution of changes in the number of functional channels, we applied ensemble fluctuation analysis to whole-cell recordings of cardiac Ca channel activity. Here we present evidence that in frog ventricular heart cells beta-adrenergic stimulation increases NF, the average number of functional Ca channels per cell. We also find that isoprenaline slows the time course of both activation and inactivation, and that the enhancement of peak current decreases gradually with greater membrane depolarization.     

Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA

There are dihydropyridine (DHP)-sensitive calcium currents in both skeletal and cardiac muscle cells, although the properties of these currents are very different in the two cell types (for simplicity, we refer to currents in both tissues as L-type). The mechanisms of depolarization-contraction coupling also differ. As the predominant voltage-dependent calcium current of cardiac cells, the L-type current represents a major pathway for entry of extracellular calcium. This entry triggers the subsequent large release of calcium from the sarcoplasmic reticulum (SR). In contrast, depolarization of skeletal muscle releases calcium from the SR without the requirement for entry of extracellular calcium through L-type calcium channels. To investigate the molecular basis for these differences in calcium currents and in excitation-contraction (E-C) coupling, we expressed complementary DNAs for the DHP receptors from skeletal and cardiac muscle in dysgenic skeletal muscle. We compared the properties of the L-type channels produced and showed that expression of a cardiac calcium channel in skeletal muscle cells results in E-C coupling resembling that of cardiac muscle.