Huge tobacco control project begun by NCI, ACS.
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American Cancer Society↗
Biomedical subjects
E Carbone
Publications and source records attributed to E Carbone.
Neuronal calcium channels: kinetics, blockade and modulation.
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Animals↗
Action of Ca2+ agonists/antagonists in mammalian peripheral neurons.
The action of several ligands on the low- (LVA,T) and high-threshold (HVA,L and N) Ca channels of adult rat sensory neurons and human neuroblastoma IMR32 cells has been investigated. In both cell types, 40 microM Cd2+ and 6.4 microM /omega-Conotoxin (omega-CgTx) selectively blocked the HVA channels, sparing the majority of LVA channels that were antagonized by amiloride and Ni2+. In 50% of the cells, however, /omega-CgTx spared also a 15% of HVA channels that proved to be sensitive to BAY K 8644. The agonistic action of BAY K 8644 on [omega-CgTx-resistant HVA channels caused a large Ba current increase, prolonged current deactivation and acceleration of HVA channels inactivation that was particularly evident in adult rat DRG.
Animals↗
Block of Na+ ion permeation and selectivity of Ca channels.
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Animals↗
Modulation of Ca channels in peripheral neurons.
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Calcium Channels↗
The relationship of modulation of major histocompatibility complex class I antigens to retrovirus transformation in rat cell lines.
The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.
Animals↗
Omega-conotoxin blockade distinguishes Ca from Na permeable states in neuronal calcium channels.
The blocking properties of the neurotoxic peptide omega-conotoxin GVIA (omega-CgTX), on neuronal Ca channels were investigated. In line with previous reports (Feldman et al. 1987; McCleskey et al. 1987), we found that micromolar concentrations of the toxin block selectively and persistently the high-threshold Ca channels of chick sensory neurons. The block by omega-CgTX could be partially relieved in low [Ca2+]o (less than 1 microM) toxin-free solutions, allowing Na ions to flow through open high-threshold Ca channels. Ca currents through these channels, however, remained permanently blocked on returning to normal Ca2+ toxin-free solutions. Also neurons which were preincubated with omega-CgTX in low Ca2+ (5 mM EGTA) failed to show high-threshold Ca currents during washing with normal Ca2+. Thus, appearance of Na currents through Ca channels in CgTX-pretreated cells was neither a consequence of unbinding of the toxin from its receptor site nor due to an interaction of EGTA with bound omega-CgTX. Na currents in CgTX-pretreated cells could be reversibly suppressed by bath applications of verapamil or by further addition of the toxin. At variance with Ca currents, block of Na currents by omega-CgTX was faster and reversible (KD 0.7 microM). Our data are consistent with the idea that neuronal Ca channels are in different conformational states when permeable to Ca2+ or Na+ ions and that omega-CgTX depresses persistently ion permeation primarily in the Ca-permeable state.
Animals↗
Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann.
The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 microM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 microM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the north American scorpion toxins. This analysis suggests that the 'fine tuning' of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).
Action Potentials↗
Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch.
1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).
Amino Acid Sequence↗
Expression of major histocompatibility complex class I antigens in normal and transformed rat thyroid epithelial cell lines.
Recent evidence suggests that the expression of abnormally high amounts of major histocompatibility complex (MHC) class I molecules may be a feature of at least some kinds of transformed cells. To investigate this aspect of neoplastic transformation we studied the expression of MHC class I antigens in an experimental model of normal, tumor-derived, and virus-transformed thyroid epithelial cell lines. The expression of MHC class I antigens has been studied by means of several monoclonal antibodies directed against either monomorphic or polymorphic epitopes and quantified by flow cytometry. Class I specific mRNA transcripts have been also analyzed by Northern blot hybridization, using a mouse genomic H-2 DNA probe. Our results indicate a modulation of MHC class I expression associated with loss of the differentiated phenotype and with transformation of thyroid epithelial cell lines. Undifferentiated cells in fact show a small quantity of these antigens, because acquisition of a fully differentiated phenotype is associated with an increase in their expression. Cell lines derived from thyroid tumors show reduced expression of MHC class I antigens, as compared to differentiated cells. Conversely, cells transformed in vitro by a retrovirus carrying the v-raski oncogene exhibit an increase in these antigens in comparison to their normal differentiated counterparts. Cells infected with a mutant virus able to transform cells only at the permissive temperature of 33 degrees C show a similar increased expression. After a shift to the nonpermissive temperature of 39 degrees C, infected cells, even those losing the transformed phenotype retain the same increased amount of MHC class I antigens. Our data suggest that the modulation of MHC class I antigen expression is strongly associated with transformation in thyroid epithelial cells.
Adenocarcinoma↗
Effect of menthol on two types of Ca currents in cultured sensory neurons of vertebrates.
The effect of menthol on voltage-dependent Ca currents was investigated in cultured dorsal root ganglion cells from chick and rat embryos. Bath application of menthol (0.1-1 mM) had different effects on the various Ca currents present in these neurons. Below -20 mV, the low threshold Ca currents were reduced in amplitude in a dose-dependent manner by menthol with little changes of their activation kinetics. In contrast to this, the time course of inactivation of the high-threshold Ca currents, activated above -20 mV from a holding potential of -80 mV, was drastically accelerated by external menthol. The action of menthol was unchanged with more positive holding potentials (-50 mV). Thus, a proposed third type of Ca current with transient activation and complete deactivation below -50 mV was either not present or not affected by menthol. Menthol exerted its action only when applied from the outside. Its effect was completely reversible within 15-20 min of wash-out. Our findings are consistent with the idea that menthol acts on two types of Ca channels coexisting on the membrane of cultured sensory neurons. Menthol blocks currents through the low voltage-activated Ca channel, and facilitates inactivation gating of the classical high voltage-activated Ca channel.
Animals↗
Blocking of the squid axon K+ channel by noxiustoxin: a toxin from the venom of the scorpion Centruroides noxius.
We have studied the selective effects of noxiustoxin (NTX), a fraction of the venom of the scorpion Centruroides noxius, on the K currents of perfused squid giant axons using the voltage-clamp technique. At concentrations below 1.5 microM, NTX blocked K currents in a voltage-independent manner, with little effect on their turning-on and turning-off kinetics. Above 1.5 microM, the block by NTX became voltage-dependent and could be partially removed by repetitive pulsing and strong depolarizations. Long repolarizations and more negative holding potentials favoured the slow restoration of channel block. Reduction of K currents by internally perfusing the fibers with solutions of low K+ concentration (200 mM), affected very little the removal of NTX-block during repetitive pulsing, suggesting that block removal depended on membrane potential and not on outward movements of K+ ions through open channels. In high extracellular K+ (300 mM) the blocking action of NTX was reduced and the instantaneous I-V characteristics showed a marked outward rectification. At 20 microM NTX, inward tail currents measured on step repolarizations to -70 mV were fully blocked, suggesting a direct interaction of the toxin with the open channel. The effects of the total venom Centruroides noxius Hoffmann was also studied. External application of 0.25 mg/ml of the venom caused a marked reduction of both Na and K currents, an effect similar to that of other scorpion venoms.
Animals↗
Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones.
1. Using the whole-cell recording mode of the patch-clamp technique, we have investigated kinetic and selectivity properties of a low-voltage-activated (l.v.a.) Ca2+ current in chick and rat dorsal root ganglion (d.r.g.) neurones. 2. L.v.a currents were activated at about -50 mV and reached maximum amplitudes between -30 and -20 mV with averages of -0.16 nA in chick and -0.3 nA in rat d.r.g. cells with 5 mM-extracellular Ca2+. Between -60 and -20 mV, the time to peak, tp, of this current decreased with increasing membrane depolarizations. An e-fold change of tp required a 14 mV potential change in chick and a 17 mV change in rat d.r.g. cells at 22 degrees C. 3. Between -50 and +20 mV inactivation of this current was fast, single exponential and voltage dependent. In rat, the time constant of inactivation, tau h, was smaller and less voltage dependent than in chick. 4. The amplitude of these currents increased by a factor of 5-10, when the extracellular Ca2+ concentration was changed from 1 to 95 mM. Amplitudes and kinetic parameters of the currents showed typical shifts along the voltage axis. No correlation between Ca2+ current amplitudes and activation-inactivation kinetics was found, suggesting that the reaction rates which control these processes are not dependent on Ca2+ entry. 5. Recovery from inactivation was voltage dependent and developed with a time constant, tau r, in the order of 1 s. tau r was nearly halved by changing the potential from -80 to -120 mV. 6. Tail currents associated with membrane repolarization were also voltage dependent and developed exponentially. Their time constant decreased by a factor of 3 when the potential was changed from -60 to -100 mV. 7. A second and more prominent Ca2+ current was activated at potentials positive to -20 mV (high-voltage-activated Ca2+ currents, h.v.a.), masking the time course of l.v.a. currents. Between -20 and 0 mV, time to peak of the entire current increased by a factor of 2 but decreased again at higher membrane potentials. Inactivation also became significantly slower in this potential range. 8. The contribution of the h.v.a. component to the total membrane current was markedly reduced using a high intracellular Ca2+ concentration, [Ca2+]i, or internal fluoride salts. This made it possible to study the kinetic parameters and the I-V characteristics of the l.v.a. current more precisely over a wider potential range (-50 to +30 mV).(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Single low-voltage-activated calcium channels in chick and rat sensory neurones.
1. Single and multiple Ca2+ channel currents were recorded from outside-out and cell-attached patches of cultured chick and rat dorsal root ganglion cells, using the patch-clamp technique. 2. Outside-out patches containing a large number of Ca2+ channels revealed Ca2+ currents resembling those from the whole cell. A low-voltage-activated (l.v.a.) and a high-voltage-activated (h.v.a.) Ca2+ current similar to those described in the accompanying paper (Carbone & Lux, 1987 b) could be distinguished. The h.v.a. current component subsided within 10 min following the formation of the patch, while the l.v.a. component lasted much longer. 3. Unitary events related to the l.v.a. Ca2+ channel could be clearly resolved in outside-out patches formed in Na+- and K+-free media containing 5-50 mM-CaCl2. 4. The amplitudes of l.v.a. channel openings were bimodally distributed, indicating the presence of two conductive states. At -40 mV, mean amplitudes of the two events were -0.29 +/- 0.07 pA and -0.47 +/- 0.085 pA in 50 mM-CaCl2, with apparent slope conductances of about 3.6 and 5.2 pS, respectively. In 5 mM-CaCl2 both slope conductances were about 3 times smaller. The mean open times were similar for both states and were fitted by a simple exponential with a time constant of about 2.5 ms at -40 mV. The time constant decreased with more-negative membrane potentials and was 0.9 ms at -100 mV. Openings frequently occurred in bursts separated by longer-lasting closures. The mean closed time during bursts was 1.33 ms at -40 mV. 5. Time and amplitude distributions of elementary events were similar for chick and rat sensory neurones and with Ba2+ and Sr2+ replacing external Ca2+. 6. In the potential range examined (from -60 to -30 mV), the first-latency distribution function revealed a distinct rise to peak which occurred at considerably earlier times than peaks of macroscopic currents. The time course of macroscopic l.v.a. Ca2+ currents could be simulated in two ways: (a) by using a five-state Markov-chain model with rate constants estimated from the transition probabilities and dwell times of the channel states, and (b) by evaluating the convolution integral of the first-latency function and the burst open probability of the channel. Both approaches suggest that activation and inactivation are weakly coupled and that the l.v.a. channel of sensory neurones reopens several times before inactivating.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Effects of dopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick.
The effects of noradrenaline and dopamine on voltage-dependent Ca currents were investigated in cultured dorsal root and sympathetic ganglion neurons from chick embryos. At concentrations of 1 to 10 microM, bath application of the neurotransmitters caused a general depression of inward Ca currents. Above -20 mV the decrease of the current amplitude was reversible and accompanied by a 2-10-fold prolongation of the activation time course. Below -20 mV, where a low voltage-activated Ca component is turned on, the size of the currents was reduced by 40% with little effect on the time course. Despite extensive wash-out, little sign of reversibility was observed in this case. Single-channel current recording in outside-out membrane patches revealed that at low membrane potentials dopamine and noradrenaline reversibly reduced single Ca-channel activity. This finding supports the view that in sensory and sympathetic neurons, both neurotransmitters affect the membrane conductance by modulating Ca permeability and not by activating catecholamine-specific channels able to carry transient outward currents. The probability of Ca channel opening is strongly reduced by addition of 10 microM of either catecholamine to the bath. The possible involvement of a voltage-dependent block of Ca channels by the neurotransmitters is discussed.
Animals↗
A low voltage-activated calcium conductance in embryonic chick sensory neurons.
Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the current's sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.
Animals↗
Selective modification of the squid axon Na currents by Centruroides noxius toxin II-10.
We have studied the effects of the purified toxin II-10, from the venom of the scorpion Centruroides noxius Hoffmann, on the Na and K currents of voltage clamped squid giant axons. Extracellular applications of 10 microM of toxin II-10 produced a selective depression of peak Na currents, with no significant effects on the time course of K currents. On pharmacologically separated Na currents, low concentrations of toxin II-10 (0.28-1 microM) caused a reversible decrease in inward and outward peak INa, with little effect on either the maintained level of the currents or their turning-off. At high concentrations (greater than 3 microM), toxin II-10 drastically reduced the peak conductance and increased both the level of the maintained conductance, and the time course of its turning-off. It is suggested that, when applied extracellularly on squid axons, toxin II-10 primarily reduces the peak Na conductance by modifying the activation of fast-inactivating Na channels. At high concentrations (10 microM), the toxin also modifies the rate constants of the transition from the inactivated to the second open state of the channel (Chandler and Meves, 1970) thus producing an increased level of the maintained Na conductance. It is also very likely, however, that peak conductance and maintained conductance reflect two separate populations of Na channels on which toxin II-10 has a differentiated action. Under these conditions, toxin II-10 would be the first reported toxin which can pharmacologically separate the two types of channels.
Amino Acid Sequence↗
High intracellular pH reversibly prevents gating-charge immobilization in squid axons.
Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332).
Animals↗