Channels In Neuroblastoma Cells Research Articles

Protective and Therapeutic Role of Ginkgo Biloba Extract Through TRPV1 Channels in an in Vitro Alzheimer's Disease Model.

The effect of Ginkgo biloba (GB) on mitochondria-dependent TRPV1 ion channels in neuroblastoma cells was investigated by creating an Alzheimer's disease (AD) model. Okadaic acid was applied on SH-SY5Y cells to create an AD model. After cellular differentiation, the study was organized with the seven main groups, examining the effect of GB on calcium depended TRPV1 channels in neuroblastoma cells AD, has been established in vitro. The higher Ca2+ concentration was detected in the GB+AD, AD and AD+GB groups when compared with the control (p<0.001). The Ca2+ level was lower in GB+AD and AD+GB groups than in the AD group (p<0.001). Also, cytosolic Ca2+ concentration was lower in the GB+AD than in the AD+GB group (p<0.05), the apoptosis and intracellular reactive oxygen species (ROS) values were higher in the GB+AD, AD and AD+GB groups than in the control (p<0.001). The apoptosis and intracellular ROS values were higher in AD group than in the GB+AD and AD+GB group (p<0.001) and the apoptosis level was higher in AD+GB group than GB+AD group (p<0.001) and the mitochondrial depolarization, caspase 3 and caspase 9 levels were higher in the GB+AD, AD and AD+GB groups when compared to the control group (p<0.001). Also, the values were lower in the GB+AD group, AD group and AD+GB groups when compared with the GB+AD+capsazepine group, AD+capsazepine group and AD+GB+capsazepine respectively (p<0.001). These results show us that GB has a protective effect besides its therapeutic effect in Alzheimer's disease via TRPV1 channel.

Potential Effects of Melatonin on TRPA1 Channels in the Prevention and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia and is defined as a progressive neurodegenerative disease. Main clinical features of AD are progressive impairment in learning and memory loss. Several studies have indicated that mitochondria play a critical role in the pathogenesis of AD. In this study, we investigated the effect of melatonin on mitochondria-dependent TRPA1 ion channels in neuroblastoma cells by creating an in vitro model of Alzheimer's disease. Okadaic acid was applied to SH-SY5Y (human neuroblastoma cell line) cells to create an AD model. After cellular differentiation, the following 7 main groups were created: Group 1 (Control), Group 2 (Mel+AD), Group 3 (Mel+AD+AP18), Group 4 (AD), Group 5 (AD+AP18), Group 6 (AD+Mel), and Group 7 (AD+Mel+AP18), and Alzheimer's disease was determined in vitro by examining the effect of melatonin on calcium-dependent TRPA1 channels in neuroblastoma cells. The Ca2+concentration was greater in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). However, there was no statistically significant difference between Mel+AD+AP18, AD+Mel+AP18 and the control. We determined that Ca2+ levels were lower in the melatonin+AD and AD+melatonin groups than in the AD group (p<0.001 and p<0.05). Additionally, cytosolic Ca2+ concentrations were found to be lower in the melatonin+AD group than in the AD+melatonin group (p<0.05). In evaluating the apoptosis and oxidative stress levels, we found that the apoptosis and intracellular ROS values were higher in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). In this respect, the mitochondrial depolarization and caspase-3 and caspase-9 levels were higher in the melatonin+AD, AD and AD+melatonin groups than in the control group (p<0.001). Additionally, the mitochondrial depolarization, caspase-3 and caspase-9 values were higher in the AD group than in the melatonin+AD and AD+melatonin groups (p<0.001), while mitochondrial depolarization and caspase-3 levels were lower in the melatonin+AD group than in the AD+melatonin group (p<0.001). However, in the same groups, there was no statistically significant difference in caspase-9 results. Additionally, the caspase-9 values were lower in the melatonin+AD group, AD group and AD+melatonin groups than in the melatonin+AD+AP18, AD+AP18 and AD+melatonin+AP18 groups, respectively (p<0.001 and p<0.05). Our results suggest that melatonin may be an effective option in the treatment and prophylaxis of Alzheimer's disease by reducing cytosolic Ca2+ concentration, apoptosis and intracellular ROS through TRPA1 channels.

Selenium enhances TRPA1 channel-mediated activity of temozolomide in SH-SY5Y neuroblastoma cells.

Neuroblastoma is a malignant solid tumor that originates from the sympathetic nervous system in early childhood. Temozolomide is used for treatment in high-risk groups with low treatment response of neuroblastomas. TRPA1 channels in neuroblastoma cells are calcium permeable channels that can be activated by reactive oxygen species (ROT). In this study, we aimed to evaluate the level of activity of temozolomide and selenium in neuroblastoma cells via TRPA1 channels. Seven main groups were formed using SH-SY5Y neuroblastoma cells. The control was divided into temozolomide (TMZ) (100μM, 24h), TMZ+SEL+AP18, SEL (sodium selenite, 100μM, 24h), and SEL+AP18 groups. Intergroup calcium signaling, intracellular reactive oxygen species, caspase-3 and caspase-9, and mitochondrial depolarization analyses were performed by channel activation with TRPA1 agonist cinnamaldehyde in all groups. Cytosolic calcium concentration, apoptosis, caspase-3 and caspase-9 activation, mitochondrial membrane depolarization, and ROT levels were higher in TMZ (p < 0.001), TMZ+SEL (p < 0.001), and SEL (p < 0.05) groups than the control group. TRPA1 was lower in TTMZ+AP18, TMZ+SEL+AP18, and SEL+AP18 groups with channel blockers than respectively TMZ, TMZ+SEL, and SEL groups without channel blockers (p < 0.05). The use of selenium with temozolomide increased the apoptotic efficacy of temozolomide via TRPA1 channels on tumor cells.

Organic Photovoltaic Sensors for Photocapacitive Stimulation of Voltage‐Gated Ion Channels in Neuroblastoma Cells

AbstractOrganic semiconductors are emerging as promising candidates for novel electrically self‐sufficient photovoltaic prosthetics for neurostimulation, especially for restoration of light sensitivity in degenerate retina. Considering future applications, it is essential to gain fundamental insight into the signaling mechanisms at the organic photosensor–electrolyte–neuron interface. Particularly, targeting voltage‐gated ion channels by a pure photocapacitive stimulation is a preferred therapeutic approach as it avoids redox reactions involved in Faradaic charge injection. The present study investigates whether single neuroblastoma (N2A) cells, grown on a photosensor based on a small molecular squaraine:fullerene photoactive layer blend, optionally covered with silicon dioxide, can be activated by photocapacitive stimulation. Indeed, upon pulsed illumination, a rapid transient photocurrent strongly depolarizes the membrane potential and subsequently activates fast‐responding voltage‐gated sodium channels. The dielectric top coating on the organic layer ensures sufficient capacitive charge injection efficiency while maintaining the rapid response of the device. Due to the high irradiance level required for photocapacitive stimulation, another slower, independent, and unintended, nonelectrical signaling pathway is identified. This activates voltage‐gated potassium channels, presumably by photothermal effects. The present study provides the basis for further improvements on standalone photovoltaic neurostimulating platforms based on organic photoactive layers.

Forty Years of Sodium Channels: Structure, Function, Pharmacology, and Epilepsy.

Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce's pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels-voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel NaV1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.

Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels

The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.

Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type), partially glycosylated (N220Q and N229Q), and unglycosylated (N220Q/N229Q) Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Inhibition of the Voltage-Gated Sodium Current and Opening of Nanopores By Ultra -Short Electric Pulses

Exposure of mammalian cells to high-voltage, ultra-short electric pulses (USEP) leads to formation of membrane nanopores and alters multiple physiological processes, including function of voltage-gated channels. However, it is not known if USEP affect the channels directly, or the effects are mediated by leak currents through nanopores and respective shifts in the transmembrane ion balance. We employed whole-cell patch-clamp to explore the effect of 300-ns USEP on voltage-gated sodium channels in neuroblastoma cells (NG108). We found that a single USEP could inhibit VG INa , with the threshold at about 1.8 kV/cm. Voltage-dependent activation and inactivation curves shifted to more negative membrane potentials: V0.5 of activation moved from −22.8±0.2mV before USEP to −26.4±0.6mV after it (mean ± s.e.), and V0.5 of inactivation changed from −65.9±0.2mV to −72.2±0.2, respectively; the slope factor did not change. Concurrently, USEP exposures induced a non-inactivating, voltage-sensitive inward current due to nanopore formation. The presence of 100μM Gd3+ in the bath buffer significantly reduced the nanopore current and also eliminated the inhibitory effect of USEP on VG INa. This finding suggests that USEP-induced inhibition of VG INa. and changes in its kinetic characteristics may be mediated by opening of nanopores and consequent alterations of the ion equilibrium.Supported by NIH (NCI) R01CA125482.

Characterization ofClostridial botulinum neurotoxin channels in neuroblastoma cells

The channel and chaperone activities of Clostridial botulinum neurotoxin (BoNT) A were investigated in Neuro 2a neuroblastoma cells under conditions that closely emulate those prevalent at the endosome. Channel activity occurs in bursts interspersed between periods of little or no activity. The channels are voltage dependent, opening only at negative voltages. Within bursts, the channel resides preferentially in the open state. The channels open to a main conductance of 105 +/- 5 pS or 65 +/- 4 pS in 200 mM CsCl or NaCl, respectively. The BoNT channels display a conspicuous subconductance of 10 +/- 2 pS. The neuroblastoma cell line appears, therefore, to be a suitable system to characterize the BoNT channel and to pursue evaluation of plausible strategies for targeted drug delivery thereby minimizing the requirement for in vivo animal testing.

Toxin-induced K+ efflux through the Na+ channel of neuroblastoma cells.

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+ influx through this channel also give rise to the efflux of 86Rb+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42K+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.

Gamma-Dendrotoxin blocks large conductance Ca 2+-activated K + channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, γ-dendrotoxin (DTX, 200 nM) blocked the outward K + current by 31.1 ± 3.5% (n = 4) with ∼500 nM Ca 2+ in the pipet solution, but had no effect on the outward K + current when internal Ca 2+ was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM γ-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca 2+ concentration was ∼500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and γ-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM γ-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. γ-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC 50 of 8.5 nM. In GH 3 cells γ-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K + currents. These results suggest that γ-DTX blocks a large conductance Ca 2+ activated K + current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca 2+ activated K + channels.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery–methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones.Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel® EMD SO3−-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil® 500-5 C18 PPN and MN SP 250/21 Nucleosil® 300-7 C18. Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant).All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na+ channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37–50).LD50 APE 2-1: 1μg/kg b.w. C. maenas (i.m.)LD50 APE 1-1: 10μg/kg b.w. C. maenas (i.m.)LD50 APE 5-3: 50μg/kg b.w. C. maenas (i.m.)

Effects of κ- and μ-opioid receptor agonists on Ca 2+ channels in neuroblastoma cells: involvement of the orphan opioid receptor

The effects of μ-, δ- and κ-opioid receptor agonists, and orphanin FQ/nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln), on K +-induced [Ca 2+] i increase were examined in SK-N-SH cells. Exposure to K + (50 mM) resulted in a [Ca 2+] i rise, which was blocked (−85%) by furaldipine (1 μM) and increased (63%) by BayK 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-carboxylate) (0.5 μM), indicating the involvement of L-type Ca 2+ channels. The κ-opioid receptor agonists 3,4-dichloro- N-Methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U-50488H) (1-50 μM) and 5,7,8- N-Methyl- N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide (U-69593) (25 μM), and the μ-opioid receptor agonist sufentanil (100 nM–3 μM) inhibited the amplitude of K +-induced [Ca 2+] i increase. The agonist of the orphan opioid receptor, orphanin FQ/nociceptin (1 μM), induced dual excitatory and inhibitory effects on the depolarisation-induced Ca 2+ influx. The effects of the opioid receptor agonists were not blocked by the κ-opioid receptor antagonist nor-binaltorphimine (1 μM), only weakly prevented by naloxone (10–100 μM) and naltrexone (100 μM), and partially prevented by pertussis toxin (100 ng/ml, 24 h). The antagonist of the orphan opioid receptor, [Phe 1Ψ(CH 2–NH)Gly 2]nociceptin(1–13)NH 2 (1 μM), prevented the inhibitory effect of U-50488H, sufentanil and orphanin FQ. The present study provides pharmacological evidence for the presence of L-type Ca 2+ channels in SK-N-SH cells, that are modulated by opioids through orphan opioid receptor activation.

Sea Anemone Peptides with a Specific Blocking Activity against the Fast Inactivating Potassium Channel Kv3.4

Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na+ channels, as well as on delayed rectifier K+ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K+ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K+ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K+ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na+ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na+ channels in neuroblastoma cells and no effect on Na+ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.

Endogenous lipids that activate cannabinoid receptors. Formation and inactivation.

The discovery of cannabinoid receptors in brain and in peripheral tissues has prompted the search for endogenous substances with cannabis-like properties. This search has led to the isolation of a lipid derivative, N-arachidonoylethanolamine (anandamide), which mimics the psychoactive effects of plant-derived and synthetic cannabinoids (Devane et al., 1992). Like Δ9-tetrahydrocannabinol, the active principle of Cannabis, anandamide binds with high affinity to cannabinoid CB1-type receptors, inhibits the twitch response in mouse vas deferens, and modulates the activities of adenylyl cyclase and voltage-dependent Ca2+ channels in neuroblastoma cells. Moreover, when administered in vivo, anandamide produces a series of behavioral responses characteristic of cannabinoid drugs (for review, see Mechoulam et al., 1994).KeywordsPhosphatidic AcidPhosphatidic AcidBioactive LipidEndogenous LipidBrain AstrocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene.

The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl- channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl- currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotonic-induced chloride conductance in neuroblastoma cells.

Alpha-latrotoxin channels in neuroblastoma cells.

The changes in ionic permeability induced by the application of alpha-latrotoxin to NG108-15 neuroblastoma x glioma cells were examined using the nystatin perforated-patch technique for whole-cell recording. Complex single channel activity appeared in the plasmalemmas after delays that ranged from 1-20 min in Krebs' solution. The conductance of a channel fluctuated among at least three broad, approximately equispaced bands, the maximum conductance being about 300 pS, and the reversal potential approximately 0 mV. The channels were permeable to Na+, K+, Ca2+ and Mg2+, poorly permeable to glucosamineH+ and Cl-, and were blocked by La3+. The channels stayed fully open in Ca(2+)-free solutions with 4 mM Mg2+, in solutions with no divalent cations and in solutions with 2 mM Ca2+ and 96 mM Mg2+. They opened infrequently if both internal and external Cl- were replaced by glutamate-. If alpha-latrotoxin opened similar channels in nerve terminals, the flux of ions through them could account for the massive release of neurotransmitter induced by the toxin.

Tetrandrine: A novel calcium channel antagonist inhibits type I calcium channels in neuroblastoma cells

Tetrandrine, an alkaloid isolated from the Chinese herb, Radix stephaniae tetrandrae, has been used clinically as a hypotensive agent for a long time. Recently, several studies have demonstrated that tetrandrine behaves like a calcium entry blocker. In the present investigation, the whole cell version of the patch clamp technique was used to study the effect of tetrandrine on the type I (transient inward) calcium current in neuroblastoma cells. These results showed that tetrandrine inhibited the transient inward current, without affecting the channel kinetics. The effects of tetrandrine were dose-dependent and reversible but did not depend on the frequency of stimulation (use-dependence) or the membrane potential. These data clearly demonstrate that tetrandrine is a novel and potent antagonist of the transient inward current in neuroblastoma cells.

Modification of K+ channel properties induced by fatty acids in neuroblastoma cells

The effects of fatty acids on voltage-dependent potassium (K+) channels in neuroblastoma cells were studied using the whole-cell current recording technique. At a concentration of 5 microM, unsaturated and medium chain length (C10-C14) saturated fatty acids accelerated the apparent inactivation of the K+ current. This effect was reversed by albumin. In the absence of exogenous fatty acids, albumin slowed the inactivation of the K+ current. The acceleration of the K+ current inactivation induced by unsaturated fatty acids was associated with an increase in the sensitivity of K+ channels to 4-amino-pyridine. It is concluded that kinetic and pharmacological properties of K+ channels are, in part, controlled by membrane fatty acids which, in this way, should contribute to an apparent diversity of K+ channels and the modulation of cell excitability.

Voltage-dependent tetrodotoxin binding to single batrachotoxin-modified Na channels recorded from intact neuroblastoma cells

To clarify the voltage-dependent actions of tetrodotoxin (TTX) on Na channels in cellular membranes, we examined the TTX block of single batrachotoxin-modified Na channels in neuroblastoma cells. We found these Na channels had a high affinity for TTX which decreased e-fold per 35.5 mV depolarization. The decrease in affinity resulted primarily from a decrease in the blocking rate for TTX; the unblocking rate increased slightly with depolarization. While the voltage-dependence of TTX binding to neuroblastoma Na channels was similar to that reported in purified Na channels incorporated in bilayers, the magnitude and voltage-dependence of the rate constants were quite different.