Spontaneous Activity of Murine Neuromuscular Junctions in the Presence of Dantrolene

By recording miniature end-plate potentials (mEPP), the effects of dantrolene (10-100 M), a blocker of ryanodine receptors, were studied on the isolated diaphragm of mice. The effects to be studied were as follows: on spontaneous secretion of acetylcholine quanta and on the pattern of interaction with ryanodine effects. Two-hour-long application of dantrolene to the muscle caused no significant changes in the amplitude and dispersion (²) of mEPP, nor on its time course. In the presence of 100 M dantrolene, the mean frequency of mEPP increased, on average, by 58.3 ± 5.9% (P < 0.05). Dantrolene suppressed in a dose-dependent manner a number of ryanodine effects (this agent was used in a concentration of 0.5 M as an intensifier of intracellular Ca²⁺ mobilization): it completely prevented the appearance of the population of high-amplitude (the so-called giant) mEPP, reduced by 50-80% the increment of the mEPP amplitude dispersion, and increased by 25-45% the mEPP mean amplitude; the above effects were induced by ryanodine application for 120 min. After preliminary application of dantrolene (10-100 M), ryanodine caused an effect not observable in the absence of dantrolene: mEPP became more frequent (140-210%). Thus, when acting on motor synapses, dantrolene behaves as a nontoxic agent, inducing only a presynaptic effect – a moderate increase in the mEPP frequency. The dual character of interaction between dantrolene and ryanodine in motor synapses was observed: on the one hand, dantrolene acts as a physiological antagonist of ryanodine by reducing the ryanodine-induced increase of dispersion and mEPP amplitude; on the other hand, dantrolene unmasks the ability of ryanodine to increase the mEPP frequency.     

Facilitation and Depression of Neuromuscular Transmission under Conditions of Blockade of Ryanodine Receptors

In our experiments on mice, end-plate currents (EPC) evoked by stimulation of the phrenic nerve were intracellularly recorded in neuromuscular synaptic junctions of the phrenic muscle. We studied the effects of a specific blocker of ryanodine receptors, ryanodine (10 to 20 M), on the amplitude and time parameters of EPC under conditions of tetanic facilitation and depression of synaptic transmission at frequencies of stimulation of 4 to 200 sec^-1. Ryanodine inhibited facilitation at stimulation frequencies of 7 to 70 sec^-1 (with maximum effect at 20 sec^-1) and accelerated depression. In the presence of ryanodine, an initial rundown of the EPC amplitude in the course of depression of transmission increased at high frequencies of stimulation (50 to 100 sec^-1), whereas the EPC amplitude at the plateau level decreased already at low frequencies (4 to 7 sec^-1). We concluded that the changes in facilitation and depression resulted from blocking of the presynaptic ryanodine receptors by ryanodine. It seems probable that calcium release from the calcium stores in murine motor terminals is a factor involved in the control of processes of transmitter secretion during short-term rhythmic activation of the junction.     

Role of Intracellular Calcium Stores on the Effect of Metabotropic Glutamate Receptors on Phosphorylation of Glial Fibrillary Acidic Protein in Hippocampal Slices from Immature Rats

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Interaction between the Cholinergic and Purinergic Control of Transmitter Release in the Neuromuscular Junction

The interaction between the cholinergic and purinergic receptors in the frog neuromuscular junction was studied using a standard microelectrode technique. The inhibitory action of an acetylcholine analog, carbachol, on transmitter release virtually disappeared when the releasing machinery was initially blocked by adenosine, indicating the existence of a functional cross-talk between the purinergic and cholinergic receptors.     

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.     

Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons

L-type voltage-sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L-type VSCCs following synaptic stimulation, using the long-wavelength Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by approximately 60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L-type VSCCs mediated approximately 50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs; approximately 15 mV) or single action potentials were much less effective at activating L-type VSCCs. Antagonist experiments indicated that activation of the NMDA-type glutamate receptor leads to a long-lasting somatic depolarization necessary to activate L-type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage-clamp pulses indicated that nuclear Ca2+ transients mediated by L-type VSCCs were produced by sustained depolarization positive to -25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non-L-type VSCCs. Our results suggest that action potentials, in combination with long-lived synaptic depolarizations, facilitate the activation of L-type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.     

Guanylyl Cyclase Participates in the Mechanism of Glutamatergic Modulation of Acetylcholine Non-Quantum Release in the Rat Neuromuscular Junction

It was shown that glutamate (10 M to 1 M) suppresses in a dose-dependent manner the non-quantum release of acetylcholine from rat motor nerve endings; the release intensity was estimated by the H effect. The action of glutamate was completely eliminated by the blockade of guanylyl cyclase by 1 M ODQ. An increase in the intracellular cGMP concentration by 1 M dibutyryl-cGMP reduced the H effect in a similar manner as glutamate did.     

Changes in Electrogenesis in the Motor Nerve Terminal with Long-Lasting High-Frequency Activation as a Factor of the Control of Transmitter Release

Using the technique of extracellular recording from the region of the neuromuscular junction in the cutaneous-sternal muscle in the frog under conditions of a reduced concentration of Ca²⁺ in the surrounding milieu, we demonstrated that long-lasting (10 min) rhythmic stimulation of the motor nerve with a frequency of 10 sec^{− 1} leads to a gradual increase in the evoked transmitter release. These changes are accompanied by a decrease in the amplitude of electrical responses of the nerve terminal (NT) and by a retardation of its second phase, as well as by a diminution of the third phase. Under conditions of long-lasting (5 min) stimulation with a frequency of 50 sec⁻¹, we observed a two-phase change in the intensity of transmitter release: on the 2nd min, the initial rise was replaced by inhibition. Modifications of the response of the NT with different stimulation frequencies were qualitatively similar, but with a frequency of 10 sec⁻¹ they were clearly expressed. Mathematical simulation of ion currents in the NT demonstrated that voltage-dependent potassium and sodium channels are inactivated in the course of long-lasting high-frequency excitation; the shape of the action potential is modified with changes in the rate of such inactivation. This leads to either an increase or a decrease of the inward calcium current. We conclude that the change in electrogenesis in the NT with long-lasting high-frequency activation of neuromuscular junctions exerts a significant influence on the dynamics of transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 108–115, March–April, 2005.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠15 min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高.     

Intracellular mechanisms participating in the formation of neuronal calcium signals

Ion and metabolic processes in the endoplasmic reticulum, mitochondria, plasma membrane, etc. providing calcium signaling in the cells of excitable and nonexcitable tissues are discussed.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 405–417, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Calcium Channels in the Nuclear Envelope of Pyramidal Neurons of the Rat Hippocampus

As is known, an increase in the concentration of Са²⁺ in the nuclei of nerve cells leads to activation of genes responsible for the formation of long-lasting postsynaptic changes; mechanisms of memory and learning are based on such changes. The pathways necessary for the entry of calcium into the nuclei of hippocampal pyramidal neurons remained unstudied. Using a patch-clamp technique, we studied what types of calcium channels exist in the membranes of isolated nuclei of pyramidal neurons of the hippocampal СА1 area. In the inner nuclear membrane of these cells, we, for the first time, found inositol trisphosphate receptors (IP₃Rs) activated by inositol trisphosphate applied in the concentration of ≥0.1 μM. The conductivity of single channels of such receptors was, on average, 366 pS; these channels were permeable for both monovalent and bivalent cations. Our data indicate that the nuclear envelope of pyramidal neurons of the hippocampal СА1 area can play the role of the calcium store from which Са²⁺ enter the cell nucleus directly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 288–292, July–August, 2008.     

Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

Caveolae position Ca_V3.2 (T‐type Ca²⁺ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca²⁺ influx to trigger Ca²⁺ sparks and large‐conductance Ca²⁺‐activated K⁺ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca²⁺ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca_v3.2 channel inhibition by Ni²⁺ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca²⁺ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca_v3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca_v1.2^?/? mice, caffeine (RyR activator) and thapsigargin (Ca²⁺ transport ATPase inhibitor), we found that sufficient SR Ca²⁺ load is a prerequisite for the Ca_V3.2‐RyR axis to generate Ca²⁺ sparks. We identified a fraction of Ca²⁺ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd³⁺ (100 µM), but insensitive to Ca_V1.2 and Ca_V3.2 channel blockade. Our data demonstrate that the VSMC Ca_V3.2‐RyR axis is down‐regulated by aging. This defective Ca_V3.2‐RyR coupling is counterbalanced by a Gd³⁺ sensitive Ca²⁺ pathway providing compensatory Ca²⁺ influx for triggering Ca²⁺ sparks in aged VSMCs.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca²⁺ action potentials when depolarized and have no detectable inward Ca²⁺ current. These fibers, however, are strictly dependent on Ca²⁺ influx for contraction, suggesting that they depend on Ca²⁺-induced Ca²⁺ release for contractile activation. The nature of the communication between Ca²⁺ channels in the sarcolemmal/tubular membrane and Ca²⁺ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr²⁺ and Ba²⁺ action potentials could be elicited in Ca²⁺-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr²⁺ or Ba²⁺ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V _max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca²⁺ concentration the channels were silent, i.e., no inward Ca²⁺ current was detected. In Ca²⁺-free solutions, inward currents carried by 138 mm Sr²⁺ or Ba²⁺ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca²⁺ channels. Peak inward current density values were low, ca.−33 μA/cm² for Sr²⁺ and −14 μA/cm² for Ba²⁺, suggesting that Ca²⁺ channels are present at a very low density. It is concluded that Ca²⁺-induced Ca²⁺ release in this crustacean muscle operates with an unusually high gain: Ca²⁺ influx through the silent Ca²⁺ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca²⁺ in the immediate vicinity of the sarcoplasmic reticulum Ca²⁺ release channels to trigger the highly amplified release of Ca²⁺ required for tension generation. Received: 5 April 1999/Revised: 15 September 1999     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function

L-type voltage-gated Ca²⁺ channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α₁-subunit comprises four repeated domains (I–IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Ca_v1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca²⁺ levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Ca_v1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α₁-subunits essential for stabilizing normal Ca²⁺ channel function.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.