Epidermal growth factor activates calcium channels by phospholipase A2/5-lipoxygenase-mediated leukotriene C4 production.

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

Auxin-induced growth and its linkage to potassium channels

This study addresses the still open question of whether or not in oily storage tissues, e.g. cotyledons of germinating rape (Brassica napus L.) seedlings' lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and the β-oxi-dation system of fatty acids are located in one or more membrane-bounded organelles. The organelles were isolated carefully and identified by marker-enzyme activities. Activities of neither lipase nor acylester acylhydrolase (EC 3.1.1) could be detected either in glyoxysomes or in mitochondria, even when various substrate emulsions were employed. Only after long-term incubations could the presence of a low lipolytic activity be demonstrated for different organellar fractions. This alkaline carboxylic ester hydrolase, whose activity is below the detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enzyme activities necessary for the conversion of saturated fatty acids to acetyl CoA was found only in the glyoxysomal cell fraction. The low β-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities unique to the mitochondrial β-oxidation system such as carnitine palmitoyltransferase (EC 2.3.1.21), carnitine acetyltransferase (EC 2.3.1.7), and acyl-CoA dehydrogenase (EC 1.3.99.3) were absent, indicating that mitochondria are not involved in fatty acid metabolism. In addition, on Western blots, antibodies raised against malate synthase (EC 4.1.3.2) and acyl-CoA oxidase (EC 1.1.3) recognized three polypeptides with molecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. Obviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and β-oxidation of fatty acids occurs exclusively in glyoxysomes. Received: 24 June 1996 / Accepted: 29 November 1996     

Platelet-derived growth factor activation of gastric mucosal calcium channels.

Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhancement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis.     

Epidermal growth factor activates calcium-permeable channels in A 431 cells

The patch clamp technique in a cell-attached configuration was used to search for calcium-permeable channels in human carcinoma A 431 cells. Unitary inward currents were recorded with 100 mM CaCl2 in a pipette, with the mean slope conductance of 2.8 pS and a reversal potential (obtained by extrapolation) of +25.5 mV. Application of epidermal growth factor (EGF) into the extracellular solution produced a transient increase in the probability of these channels being open. The effect develops with delay of about 20 s and lasts thereafter for 36 s (mean values). We propose that these channels mediate an EGF-induced increase in the concentration of cytosolic free calcium.     

Inwardly rectifying potassium channels.

Inwardly rectifying potassium (Kir) channels regulate the resting membrane potential of the cell and thereby modulate the electrical activity of cardiac and neuronal cells, insulin secretion and epithelial K(+) transport. Considerable progress in understanding the molecular structure of Kir channels and the way in which they are regulated by extracellular and intracellular modulators has been made during the past year.     

Effect of calcium ions on potassium conductivity of latrotoxin channels

It has been shown that inhibition of potassium current through latrotoxin channels by calcium ions is followed by electrostatic interaction of these ions with a total charge on the mouth of the channel.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.     

Separation of potassium and calcium channels in the nerve cell soma membranes

Investigation of isolated neurons ofHelix pomatia during intracellular dialysis revealed differences in the sensitivity of the channels for the outward potassium and inward calcium currents to changes in pH of the external medium. As a result of this difference, considerable separation of the regions of activation of the currents was obtained along the potential axis in solutions with low pH and the characteristics of the inward and outward currents could be studied during their minimal application. Channels for the outward current were shown to have some permeability for tris ions (P_Tris:P_K=0.05), which is the reason why it is impossible to block this current completely by replacing the intracellular potassium by Tris. Channels for the inward calcium current are characterized by slow inactivation, with a first-order kinetics; their momentary voltage-current characteristic curve reveals significant Goldman's rectification. The selectivity of the calcium channels for other bivalent cations is: Ba:Sr:Ca:Mg=2.8:2.6:1.0:0.2.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 6, pp. 645–653, November–December, 1978.     

Metabolic regulation and dysregulation of endothelial small conductance calcium activated potassium channels

The vascular endothelium is an important regulator of vascular reactivity and preserves the balance between vasoconstrictor and vasodilator tone during normal physiologic conditions. Example endothelial-derived vasoconstrictors include endothelin-1 and thromboxane A2; example vasodilators include nitric oxide and prostacyclin. A growing body of evidence points to the existence of a non-nitric oxide, non-prostacyclin endothelium-derived vasodilatory factor of currently unclear identity, often referred to as endothelium-derived hyperpolarizing factor (EDHF). Recent research testifies to the significance of EDHF in endothelium-dependent vascular smooth muscle relaxation. Special emphasis has been placed on the role of small conductance calcium-activated potassium channels (SK) in facilitating the endothelial and vascular responses to EDHF across the microcirculation, including coronary, mesenteric, and pulmonary vascular beds. Meanwhile, decreased activity of endothelial SK channel activity has been implicated in the pathology of a variety of disease states that alter the balance between vasodilator and vasoconstrictor tone. Hence the primary goal of this review is to characterize the physiology of endothelial SK channels in the microvasculature under normal and pathological conditions. Themes of regulation and dysregulation of SK channel activity through the action of protein kinases, reactive oxygen species, and byproducts of intermediary metabolism provide unifying principles to tie together vascular pathology in altered metabolic states ranging from hypertension to diabetes, to ischemia-reperfusion. A comprehensive understanding of SK channel pathophysiology may provide a foundation for development of new therapeutics targeting SK channels, particularly SK channel potentiators, that may have widespread application for many chronic disease states.     

A cyclic model for bimodal activation of calcium activated potassium channels in radish vacuoles.

This paper presents the mathematical framework of a cyclic model proposed for describing the transition between a fast and a slow mode (fast-slow effect) induced by the application of step membrane potentials to ion channels from radish vacuoles. A voltage stimulation pulse with frequency in the range of 2 Hz or higher increased the activation time (slow mode) of the recorded currents. When the frequency of the stimulation pattern was restored to 0.1 Hz the activation time decreased twofold (fast mode). This experimental result cannot be explained by classical kinetic theory. The model, based on a simple extension of the Hodgkin and Huxley chain, describes the whole current experimental data and provides hints on the structural conformation of ion channels.     

Calcium-activated potassium channels in chondrocytes.

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.     

Major substrate for growth factor-activated protein-tyrosine kinases is a low-abundance protein.

A scarce, soluble, conserved protein was identified as the nonphosphorylated precursor of two related 42-kilodalton phosphoproteins that contain phosphotyrosine in mitogen-stimulated but not control fibroblasts.     

Modulation of gastric mucosal calcium channels activity by platelet-derived growth factor.

A dihydropyridine-sensitive gastric mucosal calcium channels were isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The channels following labeling the calcium antagonist receptor site with [3H]PN200-100 were reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake as evidenced by La3+ displacement assays. The uptake of calcium was independent of sodium and potassium gradients indicating the electroneutral nature of the process. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microns exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhacement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channel protein showed an increase in tyrosine phosphorylation of 55 and 170 kDa calcium channel proteins. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results demonstrate the importance of PDGF in the regulation of gastric mucosal calcium uptake.     

Memory and potassium channels

The K(+)-channels of the surface membrane play a crucial role in the generation of electrical activity of a neuron. There is a large diversity of the K(+)-channels that depends on a great number (over 200) of genes encoding channels proteins. An evolutionary conservation of channel's proteins is determined. The K(+)-channels were found to have a great importance in the memory processes. It was shown on different model systems that K(+)-current of the surface membrane decreases during the learning. The antagonists of K(+)-channels were found to improve the learning and memory. It was revealed in electrophysiological experiments that K(+)-channels antagonists can either themselves induce a long-term synaptic potentiation or intensify the synaptic potentiation induced by a tetanization. The disfunction of K(+)-channels is believed to be an important link in the mechanisms of memory disturbances. In animal mutants with K(+)-channels disfunction, learning and memory are deficient. In behavioral experiments, the use of K(+)-channels openers make the learning worse. Amnesia caused by cerebral ischemia is explained by strong activity of K(+)-channels which not only inhibits neuronal excitement but also causes neurodegeneration. The question on the K(+)-channels involvement into pathophysiology of Alzheimer's disease is discussed. Neurotoxic peptide beta-amyloid, which is supposed to be involved into mechanisms of Alzheimer's disease, modulates K(+)-channels function. The effect of beta-amyloid depends on the subtype of K(+)-channels: A-channels are inhibited, and KDR-channels, on the contrary, become stronger. The effect of the cognitive enhancers (vinpocetine, piracetam, tacrine, linopirdine) on K(+)-current also depends on the subtype of K(+)-channels. Slow-inactivating K(+)-currents (IDR, IK(Ca), IM) are inhibited in the presence of these drugs, while fast-in-activating K(+)-current (A-current) remains unchanged or even increases.     

Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels

To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.