Characterization of the type of calcium channel primarily regulating GABA exocytosis from brain nerve endings

In an attempt to further characterize the type of Ca²⁺ channels primarily regulating GABA exocytosis, the effects of increasing concentrations of CTx MVIIC,--Aga IVA and other Ca²⁺ channel blockers (nitrendipine, Cd²⁺ and Ni²⁺), commonly used for pharmacologically discerning among the various types of Ca²⁺ channels, were tested on the dissected Ca²⁺ dependent fraction of the depolarization evoked release of GABA from mouse brain synaptosomes. Our results show that -CTx MVIIC inhibits GABA exocytosis with a calculated IC₅₀ of 3 M and -Aga IVA with a calculated IC₅₀ of 50 nM. The divalent cation Cd²⁺ only diminishes GABA exocytosis at 70 M, but does not modify this response at lower concentrations (i.e. 1 and 10 M). Neither nitrendipine (10 M) nor Ni²⁺ (100 M and 500 M) modified GABA exocytosis. The failure of nitrendipine at a high concentration to inhibit GABA exocytosis discards L-type Ca²⁺ channels as the main regulators of this response; likewise that of Ni²⁺ discards Ca²⁺ channels of the N-type, and the failure of nM concentrations of -CTx MVIIC or 500 M Ni²⁺, also discards alpha_1A/Q-type Ca²⁺ channels as the main regulators of the GABA response. On the basis of these results and in particular of the higher potency of -Aga IVA than -CTx MVIIC, it is concluded that the type of Ca²⁺ channels that primarily determine the exocytosis of GABA belong to a P-like type of Ca²⁺ channels.     

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide lpar;N-acetylmuramyl-L-alanyl-D-isoglutamine)

In order to understand the modification of -adrenoceptor linked signal transduction by changes in the intracellular Ca²⁺, we examined the status of -adrenoceptors (-ARs), G-proteins and adenylyl cyclase (AC) in Ca²⁺-deficiency and Ca²⁺-overload by perfusing the isolated rat heart with Ca²⁺-free medium for 5 min and Ca²⁺-containing medium for 5 min following Ca²⁺-free perfusion, respectively. Ca²⁺-depletion caused not only an increase in basal, isoproterenol-, Gpp(NH)p-, NaF- and forskolin-stimulated AC activities but also produced an increase in the ₁-AR affinity and density as well as up-regulation of G_s-protein function and uncoupling of G_i-protein to AC. Ca²⁺-repletion for 5 min following 5 min Ca²⁺-free perfusion reversed the increased AC activities to varying degrees. The ₁-AR affinity was further increased upon Ca²⁺-repletion whereas its density was decreased. Ca²⁺-repletion also decreased protein content for AC and -AR kinase but augmented the changes in G_s- and G_i-protein functions. Although low Na⁺- medium perfusion during Ca²⁺-depletion prevented the changes in G-proteins during both Ca²⁺-depletion and Ca²⁺-repletion periods, the increased ₁-AR affinity and density as well as changes in AC activities due to Ca²⁺-depletion were not affected while alterations due to Ca²⁺-repletion were fully prevented. These results suggest that changes in Ca²⁺-homeostasis may represent a mechanism for alterations in the -adrenergic signal transduction pathway in the heart under pathological conditions.     

Oxidant-induced pHi/Ca2+ changes in rat aortic smooth muscle cells: The role of atrial natriuretic peptide

The aim of this study was to investigate the effects of oxidative stress on PLD activity, [Ca²⁺]_i and pHi levels and the possible relationship among them. Moreover, since atrial natriuretic peptide (ANP) protects against oxidant-induced injury, we investigated the potential protective role of the hormone in Rat Aortic Smooth Muscle (RASM) cells exposed to oxidative stress. Water-soluble 2,2-Azobis (2hyphen;amidinopropane) dihydrochloride (AAPH) was used as free radical generating system, since it generates peroxyl radicals with defined reaction and the half time of peroxyl radicals is longer than other ROS. A significant increase of PLD activity was related to a significant decrease in pHi, while [Ca²⁺]_i levels showed an increase followed by a decrease after cell exposure to AAPH. [Ca²⁺]_i changes and pHi fall induced by AAPH were prevented by cadmium which inhibits a plasma membrane Ca²⁺ ATPase coupled to Ca²⁺/H⁺ exchanger, that operates the efflux of Ca²⁺ coupled to H⁺ influx. The involvement of PLD in pHi and [Ca²⁺]_i changes was confirmed by calphostin-c treatment, a potent inhibitor of PLD, which abolished all AAPH-induced effects. Pretreatment of RASM cells with pharmacological concentrations of ANP attenuated the AAPH effects on PLD activity as well as [Ca²⁺]_i and pHi changes, while no effects were observed with physiological ANP concentrations, suggesting a possible role of the hormone as defensive effector against early events of the oxidative stress.     

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

The effects of ₂-adrenergic stimulation on the Ca²⁺-current in mouse pancreatic -cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 M) or the ₂-adrenergic agonist clonidine (5 M) failed to reduce the Ca²⁺-current, irrespective of whether intracellular GTP (or GTP S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca²⁺-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended toincrease the Ca²⁺-current amplitude in the presence of the higher Ca²⁺-concentration. Ca²⁺-channel activation measured at 1.3 mM Ca²⁺ was not affected by clonidine. Half-maximal activation was observed at –20 mV. In addition, when Ca²⁺-currents were recorded from intact -cells, using the perforated patch whole-cell configuration, clonidine (1 M) also failed to detectably affect the Ca²⁺-current. It is therefore suggested that the inhibition of -cell electrical activity and insulin-secretion produced by ₂-adrenoreceptor stimulation does not result from suppression of the L-type Ca²⁺-current.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.     

Large-conductance ion channel measured by whole-cell voltage clamp in single cardiac cells: Modulation byβ-adrenergic stimulation and inhibition by octanol

Summary Membrane currents in single cardiac myocytes from adult guinea pigs were studied by means of the patch-clamp technique (whole-cell mode). During spontaneous or caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum openings of a novel ion channel with large unitary conductance (280 pS) can be recorded. The density of these channels and/or its open-state probability are unusually low. On average in the whole-cell mode simultaneous maximum superposition of only four channels is observed. Opening events of this channel require an intracellular Ca²⁺ transient. Activation by [Ca²⁺] _i , however, seems to be indirect; maximum opening activity occurs with a delay of several hundred milliseconds after peak [Ca²⁺] _i . Single-channel activity can be enhanced by a cyclic AMP dependent process via -adrenergic stimulation of a cell. This can also be mimicked by caffeine, most likely via inhibition of phosphodiesterase. Octanol, an inhibitor of gap-junctional coupling in a variety of tissues. causes a concentration-dependent and reversible decrease in single-channel activity. Unitary conductance is not affected by octanol. The low density of these channels in cardiac membranes and their poor selectivity render and role in normal cardiac electrical activity unlikely. A possible relation of the channel to cardiac gap junctions is discussed.     

Comparative studies on [Ca2+]i-level of fibroblasts from Alzheimer patients and control individuals

The accumulation of the -amyloid peptide (AP) in the brain, produced from the ubiquitously expressed amyloid precursor protein (APP) is a defining feature of Alzheimer's disease (AD). Consistent with studies demonstrating the importance of skin biopsy in the diagnosis of neurodegenerative disorders, we investigated whether differences in intracellular free calcium levels ([Ca²⁺]_i) of cultured cutaneous fibroblasts derived from sporadic AD patients and from age-matched control individuals might be present. [Ca²⁺]_i was measured in Fura-2AM-loaded human fibroblasts by dual wavelength spectrofluorimetry. AD cells exhibited lower [Ca²⁺]_i as compared to the control cultures. Exposure of fibroblasts to AP resulted in increased [Ca²⁺]_i of the control cells, but not of AD fibroblasts. Our test could prove useful in supporting the diagnosis of (sporadic) AD in patients suspected of suffering from the disease.     

Neurohormonal regulation of calcium in the cell

Neurohormone C (NC) is a glycopeptide isolated from bovine hypothalamus, which inhibits Ca-calmodulin (CaM)-dependent cAMP and cGMP phosphodiesterase (PDE) and is a regulator of Ca in the cell. Distribution of [⁴⁵Ca]CaCl₂ in the mitochondria and reticulum (SR) of heart and brain mitochondria and changes of Ca-binding proteins in these organelles under NC influence have been studied in the myocardium before and after isoproterenol-induced necrosis. Intraperitoneal administration of 80–100 mU of PDE inhibitory activity of NC to rats did not cause any noticeable changes in the protein content of intracellular organelles, but altered the affinity of certain proteins to⁴⁵Ca²⁺. This property of NC was especially noticable after isoproterenol necrosis. Necrotic injury of the myocardium induced Ca²⁺ storage in the mitochondria and SR of brain, and decreased the Ca²⁺ concentration in myocardial mitochondria. NC injection to the animals with necrosis was followed by Ca²⁺ release from all the studied organelles.     

α-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes

The effects of ₁ and ₂ receptor ligands on Ca²⁺/Mg²⁺-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca²⁺/Mg²⁺-ATPase, in a concentration-dependent fashion. IC₅₀ values for half-maximal inhibition for phenylephrine and clonidine were 29 M and 18 M, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca²⁺]. The present study provides biochemical evidence to support auto receptor adrenergic receptor regulation of neurotransmitter release.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.     

HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain

Background

Triptans, 5-HT_1B/ID agonists, act on peripheral and/or central terminals of trigeminal ganglion neurons (TGNs) and inhibit the release of neurotransmitters to second-order neurons, which is considered as one of key mechanisms for pain relief by triptans as antimigraine drugs. Although high-voltage activated (HVA) Ca²⁺ channels contribute to the release of neurotransmitters from TGNs, electrical actions of triptans on the HVA Ca²⁺ channels are not yet documented.

Results

In the present study, actions of zolmitriptan, one of triptans, were examined on the HVA Ca²⁺ channels in acutely dissociated rat TGNs, by using whole-cell patch recording of Ba²⁺ currents (I_Ba) passing through Ca²⁺ channels. Zolmitriptan (0.1–100 μM) reduced the size of I_Ba in a concentration-dependent manner. This zolmitriptan-induced inhibitory action was blocked by GR127935, a 5-HT_1B/1D antagonist, and by overnight pretreatment with pertussis toxin (PTX). P/Q-type Ca²⁺ channel blockers inhibited the inhibitory action of zolmitriptan on I_Ba, compared to N- and L-type blockers, and R-type blocker did, compared to L-type blocker, respectively (p < 0.05). All of the present results indicated that zolmitriptan inhibited HVA P/Q- and possibly R-type channels by activating the 5-HT_1B/1D receptor linked to G_i/o pathway.

Conclusion

It is concluded that this zolmitriptan inhibition of HVA Ca²⁺ channels may explain the reduction in the release of neurotransmitters including CGRP, possibly leading to antimigraine effects of zolmitriptan.     

Modification by hypoxia and drug treatment of cerebral ATPase plasticity

The plasticity of synaptosomal non-mitochondrial ATPases was evaluated in cerebral cortex from 3-month-old normoxic rats and rats subjected to either mild or severe intermittent normobaric hypoxia [12 hr daily exposure to N₂O₂ (9010 or 91.58.5) for four weeks]. The activities of Na⁺, K⁺-ATPase, low- and high-affinity Ca²⁺-ATPase, Mg²⁺-ATPase, and Ca²⁺, Mg²⁺-ATPase were assayed in synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The evaluations were performed after a 4-week treatment with saline (controls) or -adrenergic agents (-yohimbine, clonidine), a vasodilator compound (papaverine), and an oxygen-partial pressure increasing agent (almitrine). These treatments differently changed the adaptation to chronic intermittent hypoxia characterized by a decrease in the activity of Na⁺, K⁺-ATPase, Ca²⁺,Mg²⁺-ATPase, and high-affinity Ca²⁺-ATPase, concomitant with a modification in the activity of Mg²⁺-ATPase supported in a different way by the enzymatic forms located into the synaptosomal plasma membranes and synaptic vesicles.     

ω-Aga IVA selectively inhibits the calcium-dependent fraction of the evoked release of [3H]GABA from synaptosomes

The effect of -Aga IVA, a P-type Ca²⁺ channel blocker, on the release of the inhibitory neurotransmitter GABA and on the elevation of Ca_i induced by depolarization was investigated in [³H]GABA and fura-2 preloaded mouse brain synaptosomes, respectively. Two strategies (i.e. 20 mM external K⁺ and veratridine) that depolarize by different mechanisms the preparation were used. High K⁺ elevates Ca_i and induces [³H]GABA release in the absence of external Na⁺ and in the presence of TTX, conditions that abolish veratridine induced responses. The effect of -Aga IVA on the Ca²⁺ and Na⁺ dependent fractions of the depolarization evoked release of [³H]GABA were separately investigated in synaptosomes depolarized with high K⁺ in the absence of extermal Na⁺ and with veratridine in the absence of external Ca²⁺, respectively. The Ca²⁺ dependent fraction of the evoked release of [³H]GABA and the elevation of Ca²⁺ induced by high K⁺ are markedly inhibited (about 50%) in synaptosomes exposed to -Aga IVA (300 nM) for 3 min before depolarization, whereas the Na⁺ dependent, Ca²⁺ independent carrier mediated release of [³H]GABA induced by veratridine, which is sensitive to verapamil and amiloride, is not modified by -Aga IVA. Our results indicate that an -Aga IVA sensitive type of Ca²⁺ channel is highly involved in GABA exocytosis.     

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253

The expression of protein kinase C (PKC) isoforms and the modulation of Ca²⁺ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP₃ formation, Ca²⁺ release and Ca²⁺ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP₃ formation but inhibited the Ca²⁺ release and the Ca²⁺ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca²⁺ release induced by TG, but reduced the influx of Ca²⁺ seen in the presence of this Ca²⁺-ATPase inhibitor. These results suggest that PKC modulates elements of the IP₃/Ca²⁺ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca²⁺ channels to IP₃, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca²⁺ ATPase, and (3) modulating Ca²⁺ entry pathways.     

The effect of ATP on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) regeneration from nodal explants: association with α-tocopherol,H<Subscript>2</Subscript>O<Subscript>2</Subscript> and size of culture vessel

We investigated the effect of adenosine 5-triphosphate (ATP), -tocopherol and H₂0₂ on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants. The effect of the size of the liquid culture vessel (250-ml flask vs. 2.5-l airlift bioreactor) was also evaluated. The addition of ATP alone caused a significant increase in the number of branches (internodes) and in internodal length, but also a reduction of leaf number, compared to control cultures. It also increased significantly the accumulation of NO₃^– and K⁺ . The application of ATP + -tocopherol was associated with a significant increase in bud, internodal, leaf and petiole number, internodal, petiole and root length, as well as plant fresh weight, but reduced PO₄^3– and Ca²⁺ accumulation. The combined application of ATP + -tocopherol + H₂O₂ was associated with maximum petiole length and increased plant fresh weight but reduced Ca²⁺ accumulation. Compared to all other treatments, application of ATP + -tocopherol in bioreactor-incubated cultures produced significantly larger plants, with an increased bud number, internode lenght and soluble carbohydrate concentration, but also with a reduced fresh weight, root length and reduced NO₃^– and PO₄^3– and Ca²⁺ concentrations. These effects were associated with changes in the redox status of the regenerants, as well as dehydrogenase and peroxidase activities. The perspective for an application of ATP and antioxidants as novel, cost-efficient growth regulators in the micropropagation of this commercially important vegetable species is discussed.     

Acetyl-L-Carnitine Arginine Amide Prevents β 25–35-Induced Neurotoxicity in Cerebellar Granule Cells

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with 25–35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KC1 in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later -amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with 25–35 was able to rescue neurons from 25–35-induced neurotoxicity. We also studied the changes in Ca²⁺ homeostasis following glutamate stimulation, in control and -amyloid treated single cells, either in presence or in absence of ST857. 25–35 did not affect basal [Ca²⁺]_i, while modified glutamate-induced [Ca²⁺]_i increase, causing a sustained plateau phase of [Ca²⁺]_i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with 25–35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca²⁺ homeostasis.     

Permeation of divalent cations through α-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships

Summary -Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca²⁺, Sr²⁺ and Ba²⁺ through pores of -LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, theI-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca²⁺ and other ions in the intracellular spaces. The dependence of theI-V characteristics on voltage and Ca²⁺ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca²⁺,K=1.5m ^–1 (orK=7m ^–1 when activities are used instead of concentrations); the electrical distance of the site from the toxin-containing solution, =0.3; the free energy difference between the two barrier peaks, F =0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca²⁺ give theoretical curves that also fit the data with Sr²⁺ and Ba²⁺, indicating a low level of discrimination among these three cations.