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.     

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.     

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.     

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.     

Gating pore current in an inherited ion channelopathy

Ion channelopathies are inherited diseases in which alterations in control of ion conductance through the central pore of ion channels impair cell function, leading to periodic paralysis, cardiac arrhythmia, renal failure, epilepsy, migraine and ataxia. Here we show that, in contrast with this well-established paradigm, three mutations in gating-charge-carrying arginine residues in an S4 segment that cause hypokalaemic periodic paralysis induce a hyperpolarization-activated cationic leak through the voltage sensor of the skeletal muscle Na(V)1.4 channel. This 'gating pore current' is active at the resting membrane potential and closed by depolarizations that activate the voltage sensor. It has similar permeability to Na+, K+ and Cs+, but the organic monovalent cations tetraethylammonium and N-methyl-D-glucamine are much less permeant. The inorganic divalent cations Ba2+, Ca2+ and Zn2+ are not detectably permeant and block the gating pore at millimolar concentrations. Our results reveal gating pore current in naturally occurring disease mutations of an ion channel and show a clear correlation between mutations that cause gating pore current and hypokalaemic periodic paralysis. This gain-of-function gating pore current would contribute in an important way to the dominantly inherited membrane depolarization, action potential failure, flaccid paralysis and cytopathology that are characteristic of hypokalaemic periodic paralysis. A survey of other ion channelopathies reveals numerous examples of mutations that would be expected to cause gating pore current, raising the possibility of a broader impact of gating pore current in ion channelopathies.     

Voltage dependence of Na/K pump current in isolated heart cells

The Na/K pump usually pumps more Na+ out of the cell than K+ in, and so generates an outward component of membrane current which, in the heart, can be an important modulator of the frequency and shape of the cardiac impulse. Because it is electrogenic, Na/K pump activity ought to be sensitive to membrane potential, and it should decline with hyperpolarization. However, such voltage dependence of outward pump current has yet to be demonstrated, one reason being the technical difficulty of accurately measuring pump current over a sufficiently wide voltage range. The whole-cell patch-clamp technique allows effective control of both intracellular and extracellular solutions as well as membrane voltage. Applying this technique to myocardial cells isolated from guinea pig ventricle, we have measured Na/K pump current between -140 mV and +60 mV, after minimizing passive currents flowing through Ca2+, K+ and Na+ channels. We report here that strongly activated pump current shows marked voltage dependence; it declines steadily from a maximal level near 0 mV, becoming very small at -140 mV. Pump current-voltage relationships will provide essential information for testing models of the Na/K pump mechanism and for predicting pump-mediated changes in the electrical activity of excitable cells.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones

We have identified a serotonin-sensitive K+ channel with novel properties. The channel is active at the testing potential; its gating is moderately affected by membrane potential and is not dependent on the activity of intracellular calcium ions. Application of serotonin to the cell body or intracellular injection of cyclic AMP causes prolonged and complete closure of the channel, thereby reducing the effective number of active channels in the membrane. The closure of the channel can account for the increases in the duration of the action potential, Ca2+ influx, and transmitter release which underlie behavioural sensitization, a simple form of learning.     

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.     

Determination of the subunit stoichiometry of a voltage-activated potassium channel.

The voltage-activated K+, Na+ and Ca2+ channels are responsible for the generation and propagation of electrical signals in cell membranes. The K+ channels are multimeric membrane proteins formed by the aggregation of an unknown number of independent subunits. By studying the interaction of a scorpion toxin with coexpressed wild-type and toxin-insensitive mutant Shaker K+ channels, the subunit stoichiometry can be determined. The Shaker K+ channel is found to have a tetrameric structure. This is consistent with the sequence relationship between a K+ channel and each of the four internally homologous repeats of Na+ and Ca2+ channels.     

Alteration of ionic selectivity of a K+ channel by mutation of the H5 region

The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.     

四元含锂铷氯化物水盐体系298.2 K介稳相平衡研究

采用等温蒸发法研究了四元含锂铷氯化物体系Li+,Na+,Rb+//Cl--H2O 298.2K下的相平衡关系,测定了平衡液相的溶解度、密度和折光率.基于实验数据,绘制了该四元体系的立体图、干基图、密度-组成图和折光率-组成图.该四元体系298.2K下的介稳相图由1个共饱和点,3条单变量曲线和3个结晶区(RbCl、NaCl、LiCl·H2O)组成.将研究的结果同LiCl+KCl+RbCl+H2O体系进行了对比和分析,总结Na+和K+对三元体系Li+,Rb+//Cl--H2O的影响.应用折光率计算的经验公式对实验测定的折光率进行了验证,其最大绝对误差小于-0.0090,从而证明了实验数据的可靠性.     

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.     

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.     

Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels

Animal cells initially swell in hypotonic media by osmotic water equilibration, but their volume is subsequently regulated by a net loss of KCl and amino acids with concomitant loss of cell water. Mechanisms for regulating cell volume are important in allowing cells to adapt to variations in external tonicity and metabolic load. In red cells the KCl loss is mediated by electroneutral ion transport mechanisms. In contrast, conductive K+ and Cl- transport pathways are activated during regulatory volume decrease in several cell types including epithelia. The activation seems to be mediated by internal Ca2+, but the detailed mechanism is not known. In a leaky epithelium, the choroid plexus epithelium, we have found a cation-selective, Ca2+-permeable channel which opens with membrane stretch. The epithelium also contains a high density of the large (approximately 200 pS) type of Ca2+- voltage-activated K+ channel. Both channels are normally closed. I propose that in hypotonic media, the stretching of the cell membrane produced by the initial swelling causes influx of Ca2+ through the stretch-activated channels, which activates the neighbouring large K+ channels to produce increased K+ outflux with associated loss of cell water.     

Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel

Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.     

Intracellular ATP directly blocks K+ channels in pancreatic B-cells

It is known that glucose-induced depolarization of pancreatic B-cells is due to reduced membrane K+-permeability and is coupled to an increase in the rate of glycolysis, but there has been no direct evidence linking specific metabolic processes or products to the closing of membrane K+ channels. During patch-clamp studies of proton inhibition of Ca2+-activated K+ channels [GK(Ca)] in B-cells, we identified a second K+-selective channel which is rapidly and reversibly inhibited by ATP applied to the cytoplasmic surface of the membrane. This channel is spontaneously active in excised patches and frequently coexists with GK(Ca) channels yet is insensitive to membrane potential and to intracellular free Ca2+ and pH. Blocking of the channel is ATP-specific and appears not to require metabolism of the ATP. This ATP-sensitive K+ channel [GK(ATP)] may be a link between metabolism and membrane K+-permeability in pancreatic B-cells.     

Rapid photochemical inactivation of Ca2+-antagonists shows that Ca2+ entry directly activates contraction in frog heart

'Calcium-antagonists' are a group of pharmacological agents which are potent vasodilators and are clinically used for the treatment of angina. They are thought to block Ca2+ channels in vascular smooth muscle and myocardium but other sites of action have been proposed. These agents bind tightly to heart muscle and suppress action potential and contraction. Nifedipine and nisoldipine (BAY K 5552) are Ca2+ antagonists which have o-nitrobenzyl groups and are photolabile. We have found that short pulses of UV light rapidly inactivate these drugs in ventricular muscle. This observation allowed us to study the effect of Ca2+ antagonists on action potential, Ca2+ current and tension in conditions in which diffusion of those drugs from their site of action was not rate limiting. Our studies, described here, suggest that the primary mechanism of action of Ca2+ antagonists is the blockade of the Ca2+ channel and support the idea that extracellular space is the immediate source of contractile Ca2+ in the frog heart.     

Dihydroouabain is an antagonist of ouabain inotropic action

The Na+, K+-pump controls a wide variety of cellular systems and its inhibition by cardiac glycosides modifies important physiological functions and evokes several pharmacological effects (refs 1, 2 and refs therein). However, not all the actions of cardiac glycosides can be attributed to Na+, K+-pump inhibition and several observations show that, at low doses, cardiac glycosides stimulate the pump. It has been proposed that their positive inotropic effect could be the sum of two processes: the inhibition of the pump and a still unknown additional inotropic mechanism. In guinea pig heart, low doses of ouabain interact with high-affinity binding sites, which differ from the lower-affinity sites responsible for Na+, K+-pump inhibition. It has been suggested that ouabain interaction with these high-affinity sites could be responsible for the additional inotropic mechanism. The existence of two classes of ouabain-binding sites has been documented not only in guinea pig heart, but also in dog, rat and human heart. Dihydroouabain, a derivative of ouabain in which the lactone ring is saturated, is about 50-fold less potent than ouabain as an inhibitor of Na+, K+-pump and does not stimulate the pump at low doses. Its inotropic effect can be entirely accounted for by the inhibition of the pump. We have examined the pharmacological action of ouabain in the presence of dihydroouabain and report here that dihydroouabain reduces ouabain inotropic action but not Na+, K+-pump inhibition.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.