Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons

The antihypertensive agent mibefradil completely and reversibly inhibited T-type calcium channels in freshly isolated rat cerebellar Purkinje neurons. The potency of mibefradil was increased at less hyperpolarized holding potentials, and the apparent affinity was correlated with the degree of channel inactivation. At 35 degrees, the apparent dissociation constant Kapp was 1 microM at a holding voltage of -110 mV (corresponding to noninactivated channels) and 83 nM at a holding voltage of -70 mV (corresponding to 65% inactivation). The increased affinity was attributable mainly to a decreased off-rate. Mibefradil also inhibited P-type calcium channels in Purkinje neurons, but inhibition was much less potent. At a holding potential of -70 mV, the Kapp for mibefradil inhibition of P-type channels was approximately 200-fold higher than that for inhibition of T-type channels. Mibefradil should be a useful compound for distinguishing T-type channels from high voltage-activated calcium channels in neurons studied in vitro.     

Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.     

kappa-opioid agonist U50488 inhibits P-type Ca2+ channels by two mechanisms

The effects of U50488, kappa-opioid agonist on P-type Ca2+ channels, were studied. U50488 inhibited depolarization-induced Ca2+ uptake into rat brain synaptosomes, which was sensitive to omega-Agatoxin IVA (omega-AgaIVA; P-type Ca2+ channel blocker) and inhibited P-type Ca2+ channel currents recorded from rat cerebellar Purkinje neurons by the whole-cell patch clamp method. Dynorphin A also inhibited P-type Ca2+ channel currents. The inhibition by U50488 was biphasic; high affinity component (21%, IC50 = 8.9 x 10(-8) M) and low affinity component (79%, IC50 = 1.1 x 10(-5) M). At low concentrations of U50488 (10(-6) M), P-type Ca2+ channel current inhibition was attenuated by norbinartorphimine (nor-BNI), kappa-opioid antagonist, and by dialysis of cells with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta S). At high concentrations of U50488 (10(-5) M), P-type Ca2+ channel current inhibition was frequency-dependent. Thus U50488-induced current inhibition is mediated by two mechanisms. Its high affinity component is produced by activation of kappa-opioid receptors, whereas the low affinity component is due to its direct action on the P-type Ca2+ channel.     

Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels

In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.     

Modification of cloned brain Na+ channels by batrachotoxin

The effects of batrachotoxin (BTX) on cloned alpha-subunit Na+ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na+ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at -100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11 x 10(6) mol-1.s-1 at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The KD value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (KD = 14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between -20 mV to +50 mV. Below -30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic and stochastic properties of a persistent sodium current in mature guinea pig cerebellar Purkinje cells

Whole cell voltage-clamp techniques were employed to characterize the sodium (Na) conductances in acutely dissociated, mature guinea-pig cerebellar Purkinje cells. Three phenomenological components were noted: two inactivating and a persistent component (I(P)(Na). All exhibited similar sensitivities to tetrodotoxin (TTX; IC50 approximately 3 nM). The inactivating Na current demonstrates two components with different rates of inactivation. The persistent component activates at a more negative membrane potential than the inactivating components and shows little inactivation during a 5-s pulse. The amplitude of the persistent Na conductance had a higher Q10 than the inactivating Na conductance (2.7 vs. 1.3). (I(P)(Na) rapidly activates (approximately 1 ms) and deactivates (< 0.2 ms) and like the fast component appears to be exclusively Na permeable. (I(P)(Na) is not a "window" current because its range of activation exceeds the small overlap between the steady-state activation and inactivation characteristics of the inactivating current. Anomalous tail currents were observed during voltage pulses above -40 mV after a prepulse above -30 mV. The tails rose to a maximum inward current with a time constant of 1.5 ms and decayed to a persistent inward current with a time constant of 20 ms. The tails probably arose as a result of recovery from inactivation through the open state. The noise characteristics of (I(P)(Na) were anomalous in that the measured variance was lower at threshold voltages than would be predicted by a binomial model. The form of the variance could be partially accounted for by postulating that the maximum probability of activation of the persistent current was less than unity. The noise characteristics of (I(P)(Na) are such as to minimize noise near spike activation threshold and sharpen the threshold.     

A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells

The incretin hormones, glucagon-like peptide 1 and pituitary adenylyl cyclase-activating polypeptide, are proposed to activate a maitotoxin (MTX)-sensitive, Ca2+-dependent nonselective cation current in pancreatic beta-cells and insulinoma cells. This MTX-sensitive current is present in human beta-cells as well as in mouse and rat beta-cells, and is accompanied by a rise in cytosolic Ca2+ in voltage-clamped cells in which the activation of voltage-dependent Ca2+ channels is prevented. Activation of the nonselective cation current is inhibited by reduction of disulfide bonds with intracellular, but not extracellular, dithiothreitol, and is also abolished by intracellular dialysis with trypsin. The nonselective cation channels that carry this current have a conductance of about 30 pS, with Na+ as the major extracellular cation. We estimate that these cation channels are expressed on beta-cells at a density similar to that of ATP-sensitive potassium channels (K(ATP) channels) and exhibit spontaneous activity at basal glucose concentrations. We propose that this spontaneous cation channel activity constitutes at least part of the depolarizing background conductance that permits changes in the activity of K(ATP) channels to regulate the resting potential of beta-cells.     

Phase I and pharmacologic study of irinotecan in combination with cisplatin for advanced lung cancer

Macroscopic T-type Ca2+ currents, which are often observed in fetal and neonatal cardiac muscle cells, were not found in normal (0 of 17) adult feline ventricular myocytes. However, they were present in most (15 of 21) myocytes isolated from adult feline left ventricles with long-standing pressure-overload-induced hypertrophy. This is the first study to provide evidence in a large mammal, such as the cat, that T-type Ca2+ channels may be reexpressed in adults in association with hypertrophy resulting from slow progressive pressure overload. Importantly, this expression was stable for the duration of the hypertrophy process and was not associated with abrupt pressure overload. T-type Ca2+ currents were separated from L-type Ca2+ currents by exploiting the differences in their voltage dependence of steady-state inactivation. Depolarizations from -80 mV revealed a rapidly activating inward current that peaked in magnitude at -30 mV (-1.8 +/- 0.9 [mean +/- SD] pA/pF) and fully inactivated within 100 milliseconds in 15 of 21 hypertrophied myocytes studied. Further depolarizations activated progressively less T-type Ca2+ current, so that at +10 mV the L-type Ca2+ current predominated. In the hypertrophied myocytes that demonstrated both T-type and L-type Ca2+ currents, two distinct peaks occurred in their current-voltage relations. T-type Ca2+ currents were not evident in any of the 17 normal adult feline left ventricular myocytes studied. The purpose of T-type Ca2+ currents in hypertrophy is unclear. However, their presence may make hypertrophied myocardium more prone to spontaneous action potentials and increase the likelihood for arrhythmias in partially depolarized hypertrophied myocardium.     

Simulated responses of cerebellar Purkinje cells are independent of the dendritic location of granule cell synaptic inputs

Cerebellar Purkinje cell responses to granule cell synaptic inputs were examined with a computer model including active dendritic conductances. Dendritic P-type Ca2+ channels amplified postsynaptic responses when the model was firing at a physiological rate. Small synchronous excitatory inputs applied distally on the large dendritic tree resulted in somatic responses of similar size to those generated by more proximal inputs. In contrast, in a passive model the somatic postsynaptic potentials to distal inputs were 76% smaller. The model predicts that the somatic firing response of Purkinje cells is relatively insensitive to the exact dendritic location of synaptic inputs. We describe a mechanism of Ca2+-mediated synaptic amplification, based on the subspiking threshold recruitment of P-type Ca2+ channels in the dendritic branches surrounding the input site.     

Neuronal synchronization and ionic mechanisms for propagation of excitation in the functional network of immortalized GT1-7 neurons: optical imaging with a voltage-sensitive dye

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.     

T-type calcium channels facilitate insulin secretion by enhancing general excitability in the insulin-secreting beta-cell line, INS-1

The present study addresses the function of T-type voltage-gated calcium channels in insulin-secreting cells. We used whole-cell voltage and current recordings, capacitance measurements, and RIA techniques to determine the contribution of T-type calcium channels in modulation of electrical activity and in stimulus-secretion coupling in a rat insulin secreting cell line, INS-1. By employing a double pulse protocol in the current-clamp mode, we found that activation of T-type calcium channels provided a low threshold depolarizing potential that decreased the latency of onset of action potentials and furthermore increased the frequency of action potentials, both of which are abolished by administration of nickel chloride (NiCl2), a selective T-type calcium channel blocker. Moreover application of high frequency stimulation, as compared with low frequency stimulation, caused a greater change in membrane capacitance (deltaCm), suggesting higher insulin secretion. We demonstrated that glucose stimulated insulin secretion in INS-1 is reduced dose dependently by NiCl2. We conclude that T-type calcium channels facilitate insulin secretion by enhancing the general excitability of these cells. In light of the pathological effects of both hypo and hyperinsulinemia, the T-type calcium channel may be a therapeutic target.     

Calcium electrogenesis in neocortical pyramidal neurons in vivo

Much of what is known about Ca2+ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca2+ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca2+-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na+ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond approximately -33 mV, 71% of neurons generated high-threshold Ca2+ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K+ conductances. Consistent with this, neurons dialysed with K+ channel blockers in vivo generated high-threshold spikes that had a lower threshold (approximately -40 mV) and, with intracellular Cs+, a larger amplitude, indicating the presence of K+ currents opposing the activation of Ca2+ channels. Only 15% of cortical cells displayed low-threshold Ca2+ spikes. To compare high-threshold Ca2+ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs+, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca2+ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca2+ currents and/or the K+ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Tetrodotoxin-sensitive action potentials in smooth muscle of mouse vas deferens

Action potentials were recorded during impalements of some but not all smooth muscle cells of mouse vas deferens in response to both nerve stimulation and intracellular current injection. They were resistant to blockade by nifedipine (0.1-1.0 microM) but were blocked by tetrodotoxin (TTX, 0.2-1.0 microM) when this was added in the presence of nifedipine. It is suggested that voltage-dependent sodium (Na+) channels are present in mouse vas deferens that function to amplify calcium (Ca2+) influx through voltage-dependent Ca2+ channels.     

Clinical study on five myeloperoxidase specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) positive Churg-Strauss syndrome cases

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.     

The voltage dependence of contraction at different stimulation rates in guinea-pig ventricular myocytes

Ventricular myocytes, isolated from the guinea-pig, were stimulated to contract by 100 ms long voltage clamp pulses from -80 to 0 mV at 0.5 and 3 Hz. An increase in frequency from 0.5 to 3 Hz led to a positive inotropic effect. Contraction-voltage relationships (CVR) were determined at each frequency. The CVR at 0.5 Hz was bell shaped and peaked between 0 and +20 mV, displaying a voltage dependence similar to the L-type Ca2+ current (ICa). At 3 Hz, contractions continued to increase at positive voltages, giving a more sigmoidal CVR. At 0.5 Hz, TTX reduced the size of steady-state contractions to 91 +/- 2% of control values, but had no effect on the shape of the CVR. At 3 Hz, TTX significantly reduced (P < 0.05) the magnitude of contractions at positive voltages (> or = +20 mV) but had no significant effect on contractions at voltages negative to 0 mV. These data illustrate that intracellular sodium activity (aNa(i)) and, in particular, Na+ entry due to the sodium current (INa) are important in determining the voltage dependence of contraction at positive voltages. Thapsigargin (2.5 microM), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced the size of steady-state contractions at 0 mV to 65 +/- 7% at 0.5 Hz. Increasing frequency to 3 Hz abolished the positive inotropy seen under control conditions. With thapsigargin present, contractions at 0.5 Hz were reduced at all potentials and the CVR was bell shaped. At 3 Hz the CVR was sigmoidal in shape. Contractions were significantly inhibited by thapsigargin at all potentials, but most significantly at more positive potentials (> or = +20 mV). These data show that, at normal body temperature, the shape of the CVR of guinea-pig ventricular myocytes changes with stimulation rate. Due to the voltage dependence of ICa, contractions evoked at positive voltages at 3 Hz must be supported by other mechanisms. The sensitivity of such contractions to TTX and thapsigargin suggests the involvement of both a Na(+)-dependent process and the SR. One possibility is that when aiNa and the Ca2+ content of the SR are raised at higher stimulation rates, enhanced Ca2+ entry via reverse Na(+)-Ca2+ exchange leads to a direct activation of the myofilaments and, to a lesser extent, the release of Ca2+ from the SR.     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones. 2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1-100 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, which was 5 fold less than the value obtained at -90 mV. Repeated activation of TTX(R) by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 microM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX(R) and markedly slowed the recovery from channel inactivation. 4. Fast TTX-sensitive Na+ currents (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 4030W92 was 20+/-6% (n=6). Low threshold T-type Ca2+ currents were inhibited by 17+/-8% and 43+/-3% by concentrations of 4030W92 of 30 microM and 100 microM, respectively (n=6). 6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10-30 microM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7+/-1.5 to 4.2+/-1.0 and 2.6+/-1.1 in the presence of 10 microM and 30 microM 4030W92, respectively (n=5). 7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na+ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na+ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.     

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.     

Voltage- and ligand-gated ion channels in floor plate neuroepithelia of the rat

Whole-cell patch-clamp recordings were used to characterize the membrane properties and ion channel complement of floor plate neuroepithelia in embryonic and neonatal rats. The average resting potential was close to -60 mV, the capacitance was approximately 7 pS and the membrane time constant averaged 31 ms, in both neonates and embryos. Two types of K+ current were identified (i) a slowly activating, slowly inactivating current that was present in all cells, and (ii) a rapidly inactivating current that was present in 39% of cells from neonates and 64% of cells from embryos. K+ currents were significantly larger in neonates than embryos. Na+ currents were absent from all neuroepithelial cells examined. In contrast, the majority of floor plate cells exhibited a significant Ca2+ current. Biophysically this current activated at potentials positive to 60 mV and exhibited fast, voltage-dependent, inactivation. The Ca2+ current was equipermeant to Ca2+ and Ba2+, sensitive to 40-120 microM Ni2+ and only slightly inhibited by 100 microM Cd2+. These and other observations indicated this current is mediated by low-voltage-activated (i.e. T-type) Ca2+ channels. The majority of floor plate cells tested also exhibited responses to the neurotransmitter GABA which produced robust inward currents at negative membrane potentials, in chloride-loaded cells. Both the pharmacology and voltage-dependence of the GABA-activated currents indicated they arose from activation of GABA(A) receptors.     

Synaptically activated low-threshold muscarinic inward current sustains tonic firing in rabbit prevertebral sympathetic neurons

Whole-cell patch-clamp experiments were performed on non-dissociated rabbit coeliac sympathetic neurons in the presence of nicotinic blockers. Coeliac neurons were classified as either silent or spontaneously active (pacemaker) cells. Under voltage-clamp conditions, pacemaker cells exhibited a steady-state N-shaped current-voltage relationship due to the presence of a persistent voltage-dependent inward current in the potential range of -100 to approximately -20 mV. This inward current sustained the regular firing activity of pacemaker cells and was absent from quiescent neurons. It disappeared in the presence of tetrodotoxin and in low Ca(2+)-high Mg2+ external solutions and was enhanced by eserine. Splanchnic nerve stimulation induced slow regenerative depolarizations and firing discharges in silent neurons by activating a low-threshold voltage-sensitive inward current. The synaptic current had a U-shaped voltage-dependence from -96 to approximately -20 mV and exhibited the dynamic properties of the muscarinic voltage-dependent inward current INa,M. It gave the current-voltage relationship an N shape similar to that observed in spontaneously active cells. The muscarinic antagonists atropine and pirenzepine abolished the inward current present in pacemaker cells and that induced by nerve stimulation in silent neurons. These data provide evidence that both spontaneous firing activity and nerve-evoked depolarizing responses in coeliac neurons are sustained by the activation of the muscarinic Na,M current. The tonic activation of INa,M in spontaneously firing cells results from a sustained Ca(2+)-dependent tetrodotoxin-sensitive release of acetylcholine. This study provides evidence that the role of the muscarinic receptors is not purely a neuromodulatory one, but that these receptors are directly involved in ganglionic neurotransmission.