Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.     

Altered regulation of airway epithelial cell chloride channels in cystic fibrosis

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Restoration by calmodulin of a Ca2+-dependent K+ current missing in a mutant of Paramecium

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.     

Voltage-dependent calcium channels from Paramecium cilia incorporated into planar lipid bilayers

Two different divalent cation-selective channels from Paramecium cilia were incorporated into planar lipid bilayers. Both channels were much more permeable to divalent than univalent cations, and one of them discriminated significantly among the divalent cations. The selectivity and voltage dependence of the latter channel are comparable to those of voltage-dependent calcium channels found in a variety of cells. A combined biochemical, biophysical, and genetic study of calcium channels is now possible.     

Ion channels in yeast

Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

Atrotoxin: a specific agonist for calcium currents in heart

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.     

External calcium ions are required for potassium channel gating in squid neurons

The effects of calcium removal on the voltage-dependent potassium channels of isolated squid neurons were studied with whole cell patch-clamp techniques. When the calcium ion concentration was lowered from 10 to 0 millimolar (that is, no added calcium), potassium channel activity, identified from its characteristic time course, disappeared within a few seconds and there was a parallel increase in resting membrane conductance and in the holding current. The close temporal correlation of the changes in the three parameters suggests that potassium channels lose their ability to close in the absence of calcium and simultaneously lose their selectivity. If potassium channels were blocked by barium ion before calcium ion was removed, the increases in membrane conductance and holding current were delayed or prevented. Thus calcium is an essential cofactor in the gating of potassium channels in squid neurons.     

Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells

Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.     

Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum

The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.     

Verapamil和CPZ对金冠苹果衰老生理的影响

对金冠苹果经Ca2 通道阻塞剂Verapamil,CaM拮抗剂Chloropromaize（CPZ）处理后的一些生理生化变化做了研究。结果表明,Verapamil和CPZ促进果肉软化,增加膜透性和可溶性蛋白质含量,分别提早呼吸跃变、提高呼吸速率的峰值,并且加大了过氧化物酶和超氧化物歧化酶活性的变化幅度。     

IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels

Lambert-Eaton syndrome, an autoimmune disorder frequently associated with small-cell carcinoma of the lung, is characterized by impaired evoked release of acetylcholine from the motor nerve terminal. Immunoglobulin G (IgG) antibodies from patients with the syndrome, applied to bovine adrenal chromaffin cells, reduced the voltage-dependent calcium channel currents by about 40 percent. When calcium was administered directly into the cytoplasm, however, the IgG-treated cells exhibited normal exocytotic secretion, as assayed by membrane capacitance measurement. Measurement with the fluorescent calcium indicator fura-2 indicated that the IgG treatment reduced potassium-stimulated increase in free intracellular calcium concentration. The pathogenic IgG modified neither kinetics of calcium channel activation nor elementary channel activity, suggesting that a reduction in the number of functional calcium channels underlies the IgG-induced effect. Therefore, Lambert-Eaton syndrome IgG reacts with voltage-dependent calcium channels and blocks their function, a phenomenon that can account for the presynaptic impairment characteristic of this disorder.     

Identification of an alpha subunit of dihydropyridine-sensitive brain calcium channels

Voltage-sensitive calcium channels in different tissues have diverse functional properties. Polyclonal antibodies (PAC-2) against the alpha subunits of purified rabbit skeletal muscle calcium channels immunoprecipitated calcium channels labeled with the dihydropyridine PN200-110 from both skeletal muscle and brain. The immunoreactivity of PAC-2 with the skeletal muscle channel was greater than that with the brain calcium channel and was absorbed only partially by prior treatment with the brain channel. PAC-2 specifically recognized a large peptide in synaptic plasma membranes of rabbit brain with an apparent molecular size of 169,000 daltons. This protein resembles an alpha subunit of the skeletal muscle calcium channel in apparent molecular weight, antigenic properties, and electrophoretic behavior after reduction of disulfide bonds. Thus, the dihydropyridine-sensitive calcium channel of rabbit brain has an alpha subunit that is homologous, but not identical, to those of the skeletal muscle calcium channel. The different functional properties of these two calcium channels may result from minor variations in structurally similar components.     

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.     

Atrionatriuretic peptide transforms cardiac sodium channels into calcium-conducting channels

The atrionatriuretic peptide (ANP) is released from atrial cells in response to increased extracellular fluid volume and reduces sodium absorption by the kidney, thus reducing the blood volume. In this report, ANP suppressed the calcium and sodium currents in rat and guinea pig ventricular myocytes. The suppression of sodium current was caused by enhanced permeability of the sodium channel to calcium without significant changes in the kinetics or the tetrodotoxin sensitivity of the channel. Thus, ANP may regulate the sodium channel by altering its cationic selectivity site to calcium, thereby repressing the sodium current. The suppression of sodium and calcium channels and the resultant depressed excitability of the atrial cells may help to regulate ANP secretion.     

Beta-adrenergic inhibition of cardiac sodium channels by dual G-protein pathways

The signaling pathways by which beta-adrenergic agonists modulate voltage-dependent cardiac sodium currents are unknown, although it is likely that adenosine 3'5'-monophosphate (cAMP) is involved. Single-channel and whole-cell sodium currents were measured in cardiac myocytes and the signal transducing G protein Gs was found to couple beta-adrenergic receptors to sodium channels by both cytoplasmic (indirect) and membrane-delimited (direct) pathways. Hence, Gs can act on at least three effectors in the heart: sodium channels, calcium channels, and adenylyl cyclase. The effect on sodium currents was inhibitory and was enhanced by membrane depolarization. During myocardial ischemia the sodium currents of depolarized cells may be further inhibited by the accompanying increase in catecholamine levels.     

Permeation of calcium ions through non-NMDA glutamate channels in retinal bipolar cells.

The conduction of calcium ions through glutamate-gated channels is important in the induction of long-term potentiation and may trigger other cellular changes. In retinal bipolar cells, which lack the N-methyl-D-aspartate (NMDA) type of glutamate-gated channel, calcium permeability through non-NMDA channels was examined. Changes in extracellular calcium concentration unexpectedly affected the reversal potential for glutamate-induced currents in a manner consistent with these channels being highly permeable to calcium. External magnesium ions promote desensitization of these non-NMDA channels in a voltage-independent way. Thus, in addition to non-NMDA channels that conduct only sodium and potassium, there is a class that is also permeable to calcium.     

Regulation of a heart potassium channel by protein kinase A and C

The enzymes adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C regulate the activity of a diverse group of cellular proteins including membrane ion channel proteins. When protein kinase A was stimulated in cardiac ventricular myocytes with the membrane-soluble cAMP analog 8-chlorphenylthio cAMP (8-CPT cAMP), the amplitude of the delayed-rectifier potassium current (IK) doubled when recorded at 32 degrees C but was not affected at 22 degrees C. In contrast, modulation of the calcium current (ICa) by 8-CPT cAMP was independent of temperature with similar increases in ICa occurring at both temperatures. Stimulation of protein kinase C by phorbol 12,13-dibutyrate also enhanced IK in a temperature-dependent manner but failed to increase ICa at either temperature. Thus, cardiac delayed-rectifier potassium but not calcium channels are regulated by two distinct protein kinases in a similar temperature-dependent fashion.     

Early enhancement of calcium currents by H-ras oncoproteins injected into Hermissenda neurons

Influx of calcium through membrane channels is an important initial step in signal transduction of growth signals. Therefore, the effects of Ras protein injection on calcium currents across the soma membrane of an identified neuron of the snail Hermissenda were examined. With the use of these post-mitotic cells, a voltage-sensitive, inward calcium current was increased 10 to 20 minutes after Harvey-ras oncoproteins were injected. The effects of oncogenic Harvey ras p21 protein (v-Ras) occurred quickly and were sustained, whereas the effects of proto-oncogenic ras protein (c-Ras) were transient. This relative potency is consistent with the activities of these oncoproteins in stimulating cell proliferation. Thus, this calcium channel may be a target for Ras action.