Novel mechanism of voltage-dependent gating in L-type calcium channels

Activation of voltage-dependent calcium channels by membrane depolarization triggers a variety of key cellular responses, such as contraction in heart and smooth muscle and exocytotic secretion in endocrine and nerve cells. Modulation of calcium channel gating is believed to be the mechanism by which several neurotransmitters, hormones and therapeutic agents mediate their effects on cell function. Here we describe a novel type of voltage-dependent equilibrium between different gating patterns of dihydropyridine-sensitive (L-type) cardiac Ca2+ channels. Strong depolarizations drive the channel from its normal gating pattern into a mode of gating characterized by long openings and high open probability. The rate constants for conversions between gating modes, estimated from single channel recordings, are much slower than normal channel opening and closing rates, but the equilibrium between modes is almost as steeply voltage-dependent as channel activation and deactivation at more negative potentials. This new mechanism of voltage-dependent gating can explain previous reports of activity-dependent Ca2+ channel potentiation in cardiac and other cells and forms a potent mechanism by which Ca2+ uptake into cells could be regulated.     

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.     

Reversible uncoupling of inactivation in N-type calcium channels.

N-type calcium channels are thought to be expressed specifically in neuronal cells and to have a dominant role in the control of neurotransmitter release from sympathetic neurons. But their unitary properties are poorly understood and the separation of neuronal Ca2+ current into components carried by N-type or L-type Ca2+ channels is controversial. Here we show that individual N-type Ca2+ channels in sympathetic neurons can carry two kinetically distinct components of current, one that is rapidly transient and one that is long lasting. The mechanism that gives rise to these two components is unexpected for Ca2+ channels: a test depolarization elicits either a rapidly inactivating, single short burst with an average duration of 40 ms, or sustained, noninactivating channel activity lasting for over 1 s. The switching between inactivating and noninactivating activity is a slow process, the occurrence of each type of unitary kinetic behaviour remaining statistically correlated over several seconds. Variable coupling of inactivation in N-type Ca2+ channels could be an effective mechanism for the modulation of neuronal excitability and synaptic plasticity.     

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.     

The agonist effect of dihydropyridines on Ca channels

Dihydropyridines (DHP) have great potential for clinical use because of their inotropic and vasomotor effects. The mechanism of action is unknown although Ca currents have been implicated. Here we report measurements of single channel and whole cell cardiac Ca currents after application of two DHP agonists BAY K 8644 and CGP 28 392. Whole cell Ca currents from individual myocytes were increased and the 50% effective doses (ED50) were similar to those reported for contractility in rabbit aorta and guinea pig heart and catecholamine release from cat adrenal glands. The measured ED50 was also consistent with the apparent dissociation constant (Kd) of a high affinity binding site present in cardiac sarcolemmal vesicles. We propose that the molecular basis for these results is an increase in the probability that a single Ca channel, having opened and closed, will subsequently re-open during membrane depolarization. At high concentrations of BAY K 8644 and in the presence of 96 mM Ba, different effects are observed, primarily a marked prologation of open time.     

Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers?

Endothelial cells line the inner surface of blood vessels and act as the main barrier to the passage of cells and large molecules from the blood stream to the tissues. Recent interest in the part played by the endothelium in regulating vascular tone has focused on the synthesis and secretion of prostacyclin and an endothelium-derived relaxing factor. Endothelial cells respond to blood-borne agonist, but how the endothelium senses and responds to mechanical forces generated by the flow of blood under pressure is not known. Here we report patch-clamp recordings of ion channel activity from cell-attached membrane patches on aortic endothelial cells. In most of the patches examined, we observed unitary inward currents associated with the opening of a cation-selective channel (approximately 40 pS in standard saline). The channel is permeable to Ca2+ and its opening frequency increases when the membrane is stretched by applying suction through the patch electrode. The presence of mechanotransducing ion channels in endothelial cells may help explain how the endothelium mediates vascular responses to haemodynamic stresses.     

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.     

Three types of neuronal calcium channel with different calcium agonist sensitivity

How many types of calcium channels exist in neurones? This question is fundamental to understanding how calcium entry contributes to diverse neuronal functions such as transmitter release, neurite extension, spike initiation and rhythmic firing. There is considerable evidence for the presence of more than one type of Ca conductance in neurones and other cells. However, little is known about single-channel properties of diverse neuronal Ca channels, or their responsiveness to dihydropyridines, compounds widely used as labels in Ca channel purification. Here we report evidence for the coexistence of three types of Ca channel in sensory neurones of the chick dorsal root ganglion. In addition to a large conductance channel that contributes long-lasting current at strong depolarizations (L), and a relatively tiny conductance that underlies a transient current activated at weak depolarizations (T), we find a third type of unitary activity (N) that is neither T nor L. N-type Ca channels require strongly negative potentials for complete removal of inactivation (unlike L) and strong depolarizations for activation (unlike T). The dihydropyridine Ca agonist Bay K 8644 strongly increases the opening probability of L-, but not T- or N-type channels.     

Sarcoplasmic reticulum contains adenine nucleotide-activated calcium channels

Rapid calcium efflux from the sarcoplasmic reticulum (SR) is a necessary step in excitation-contraction coupling in skeletal muscle and is thought to be mediated by a calcium channel. Calcium efflux has been studied in fragmented SR vesicles by radioisotope efflux and fluorescence measurements. Several laboratories have reported that adenine nucleotides can stimulate calcium efflux from SR. In recent reports, Ca2+ release with a first-order rate constant as high as 100 s-1 has been observed for nucleotide-stimulated Ca2+ release from SR vesicles. Also, radioisotope efflux was blocked by Mg2+ and micromolar concentrations of the polycationic dye, ruthenium red. These high rates of transport are difficult to reconcile with a mechanism other than passive diffusion through a nucleotide-activated 'calcium release channel'. Using the fusion technique for inserting SR proteins into planar lipid bilayers, we report here single-channel recordings of calcium release channels from purified 'heavy' SR membranes. Channels have been identified on the basis of their activation by adenine nucleotides, blockade by ruthenium red, and selectivity for divalent cations. Surprisingly, the channel studied here exhibits an unusually large conductance of 170 pS in 50 mM Ba2+ while still being capable of discriminating against monovalent cations by a permeability ratio, P(Ba)/P(Cs) = 11.4.     

Effects of protein kinase C activators on cardiac Ca2+ channels

Phorbol esters have marked effects on voltage-dependent Ca2+ channels. Inhibitory and stimulatory effects on cardiac Ca2+ channels have been attributed in both cases to activation of protein kinase C. We show that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate stimulates dihydropyridine-sensitive 45Ca2+ influx in primary cultures of neonatal rat ventricular myocytes within 5 s, but that after a 20-min pre-incubation period the phorbol ester markedly inhibits 45Ca2+ influx. The sequence of stimulation followed by inhibition is confirmed in cell-attached patch clamp recordings of single Ca2+ channel currents. The stimulatory effect is faster at 0 mV than at -40 mV, leading to the novel conclusion that the rate of protein kinase C activation is modulated by the state of the Ca2+ channel.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane

The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.     

心力衰竭对QT间期变异性的影响

探讨QT间期变异性在心力衰竭中的意义。对17例心力衰竭患者、22例无心力衰竭者进行动态心电图检查，并进行QT间期动态分析。结果显示，心力衰竭组的QT间期变异性（QTV）明显大于无心力衰竭组，心力衰竭影响QT间期变异性。     

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.     

Calcium entry through stretch-inactivated ion channels in mdx myotubes.

Recent advances in understanding the molecular basis of human X-linked muscular dystrophies have come from the identification of dystrophin, a cytoskeletal protein associated with the surface membrane. Although there is little or virtually no dystrophin in affected individuals, it is not known how this causes muscle degeneration. One possibility is that the membrane of dystrophic muscle is weakened and becomes leaky to Ca2+. In muscle from mdx mice, an animal model of the human disease, intracellular Ca2+ is elevated and associated with a high rate of protein degradation. The possibility that a lack of dystrophin alters the resting permeability of skeletal muscle to Ca2+ prompted us to compare Ca2(+)-permeable ionic channels in muscle cells from normal and mdx mice. We now show that recordings of single-channel activity from mdx myotubes are dominated by the presence of Ca2(+)-permeable mechano-transducing ion channels. Like similar channels in normal skeletal muscle, they are rarely open at rest, but open when the membrane is stretched by applying suction to the electrode. Other channels in mdx myotubes, however, are often open for extended periods of time at rest and close when suction is applied to the electrode. The results show a novel type of mechano-transducing ion channel in mdx myotubes that could provide a pathway for Ca2+ to leak into the cell.     

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.     

Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.     

Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel

Many excitable cells contain at least two different voltage-dependent Ca channels (L- and T-type). The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation, which increases the probability of it being open, and is readily blocked by Ca channel blockers including dihydropyridines and phenylalkylamines. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo, that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules contains three peptides of relative molecular mass (Mr) 142,000 (142K), 56K and 31K. The cAMP kinase incorporates one mol phosphate per mol of the 142K peptide and binding of (+)PN-200/110, a potent Ca antagonist, is allosterically affected by D-cis-diltiazem and verapamil. The purified dihydropyridine-receptor complex has also been incorporated into phospholipid bilayer membranes. Here, we show for the first time that the complex can be reconstituted to form a functional 20-pS Ca channel that retains the principal regulatory, biochemical and pharmacological properties of membrane-bound L-type Ca channels.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.