Types Of Voltage-dependent Calcium Channels Research Articles

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Calcium modulation of the kinetics of evoked quantum secretion in neuromuscular synapses of cold- and warm-blooded animals

Calcium entry into the nerve endings through voltage-dependent calcium channels triggers a chain of events leading to exocytosis of neurotransmitter, providing the transmission of excitation through the synapse. In this regard, a significant role of calcium ions and presynaptic calcium channels in the modulation of secretion is evident. However, the question of the contribution of different types of voltage-dependent calcium channels in the calcium regulation parameters of the quantal secretion still remains unclear. The secretion kinetics characterizes a degree of synchrony of the neurotransmitter release. In recent decades it is regarded as one of the important factors maintaining the effectiveness of the synaptic transmission. Since neuromuscular synapses of frogs and mice are classical objects of physiological and pharmacological studies, the results of which are summarized and extrapolated to other synapses, it is interesting to compare changes of the acetylcholine secretion in these synapses under different conditions of calcium entry into the nerve endings. In this review we discuss the data on the neuromuscular synapses of frogs and mice and analyze some aspects of calcium regulation and involvement of different types of voltage-dependent calcium channels in the modulation of the acetylcholine secretion kinetics.

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Cav2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Cav1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Cav3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar β-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein–protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.

Presynaptic voltage-dependent calcium channels and regulation of the transmitter release in the peripheral nervous system

Calcium ions entering into the nerve terminal through voltage-dependent calcium channels during action potential initiate neurotransmitter release and thereby play an important role in the transmission of excitation. In addition to the amount of released neurotransmitter quanta (the quantal content of the endplate potential), another important characteristic of exocytosis is the kinetics of release of individual quanta that form total multiquantal response. Previously, we showed the dependence of the temporal parameters of neurotransmitter quantum release on the extracellular calcium ion concentration, but it remained unclear which calcium channel types are involved in the modulation of the quantal content and release kinetics. It is commonly accepted that, in contrast to the central nervous system, wherein the secretion take part various types of voltage-dependent calcium channels, exocytosis in the peripheral nervous system of adult animals mainly occurs owing to one type of calcium channels. This is the P/Q type (Ca V 2.1 in the latest classification) blocked with FTX toxin and ω -agatoxin IVA in mammalian neuromuscular junction. In frog synapses, this is the N type channels (Ca V 2.2), which are blocked by the ω -conotoxin GVIA. At the same time, the ability of FTX, a specifc P/Q channel blocker, to decrease the quantal content in frog synapses and effects of dihydropyridines that block L type (Ca V 1.2) channels in the developing Xenopus synapses, and L type (Ca V 1.2) channels activator effects in frog synapses, taken together, indicate the possibility of participation of other types of calcium channels in the exocytosis regulation in peripheral synapses. The possible role of changes in the activity of not only P/Q channels, but also other calcium channel types, in modulation of the temporal parameters of neurotransmitter release was shown in peripheral synapses of mouse and crayfish. In order to identify the type of voltage-dependent calcium channels modulating of the quanta secretion in frog and mouse synapses, the effects of specific blockers of various channel types on the endplate potentials quantal content and transmitter release kinetics were analyzed. On isolated Rana ridibunda neuromuscular preparation of m. cutaneus pectoris and on the mouse diaphragm the evoked uniquantal endplate currents were registered by the extracellular electrodes and the fluctuations of the real synaptic latency were estimated. Blocking of the P/Q-, N-, and L-channels in nerve-muscle junction of the frog by the appropriate specific blockers led to a decrease in the amount of released quanta, as well as in the synaptic latency variance, that indicate the synchronization of the process of synaptic vesicles exocytosis. Obtained results indicate that in low-calcium solution the kinetics of quanta secretion in the frog synapse may be regulated by N-, P/Q-, and L-calcium channels. In the mouse nerve muscle junctions at low calcium concentration and infrequent stimulation, when asynchrony of the secretion was more pronounced, the block of the P/Q-channels led to a decrease in the amount of released quanta without changing the kinetics of their secretion, while blocking the L-type channels only decreased variance of synaptic latency. The fact that the number of released quanta and the time course of their secretion may change independently when various types of voltage-dependent calcium channels are blocked witnesses that the role of N-, P/Q-, and L-channels in modulation of the process of synaptic vesicles exocytosis is different.

Voltage‐dependent calcium channels in human renal arteries

Several types of voltage‐dependent calcium channels (VDCC) Cav1.2 (L‐type), Cav2.1 (P/Q‐ type) and Cav3.1/ Cav3.2 (T‐ type) are expressed in vascular smooth muscle cells from rodents and are important for renal resistance. We examined whether human renal arteries express several types of VDCC and assessed their functional importance. The expression of Cav 1.2, mRNA was demonstrated in all human renal and interlobular arteries tested whereas Cav 2.1, Cav 3.1 and Cav 3.2 only were observed in some patients. Immunolabeling revealed expression of Cav1.2 and Cav2.1 in human arteries. Using a myograph set‐up, the functional importance of different types of VDCC was investigated in freshly isolated human interlobular arteries. High K+ led to a dose‐dependent constriction of the arteries. Nifedipine blocked the K+ (70 mM) induced constriction with an EC50 of 1.2 × 10−8 M. Blockade of T‐type channels by mibefradil inhibited the constriction with an EC50 of 6.8 × 10−7 M. The response was very variable between individual patients. Blocking P/Q‐type channels using ω‐agatoxin IVA (10−8 nmol/L) had no significant effect (14.8 ± 5.0 % decrease) on the constriction.We conclude that all human renal arteries express functionally significant L‐type, VDCC while there is a more variable presence and significance of P/Q‐ and T‐type VDCC.Research Support: Lundbeck Foundation, Danish Medical Research Council, Danish Heart Foundation

Participation of different types of voltage-dependent calcium channels in evoked quantal transmitter release in frog neuromuscular junctions

Participation of different types of voltage-dependent calcium channels in evoked quantal transmitter release in frog neuromuscular junctions

Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel α 1 subunits in the dorsal horn of spinal cord in mice

To understand better which voltage-dependent calcium channels (VGCCs) are involved in nociceptive neurotransmission, we investigated the pharmacological properties and distribution of VGCCs in the mouse spinal cord. A behavioral assay revealed that intrathecal injections of ω-agatoxin TK, ω-agatoxin IVA, ω-conotoxin GVIA, and SNX-482, which block P/Q-, P/Q-, N-, and R-type calcium channels, respectively, produced analgesic effects, while an L-type channel blocker had no such effect. An electrophysiological study demonstrated the presence of various types of VGCCs within dorsal root ganglion (DRG) neurons. Immunohistochemistry revealed distinct localization of P/Q-, N-, L-, and R-type calcium channel subunits to the dorsal horn of the spinal cord. The results of this study revealed the localization and functions of several calcium channels that are involved in nociceptive neurotransmission within the dorsal horn of the mouse spinal cord.

Differential expression of α 1 and β subunits of voltage dependent Ca 2+ channel at the neuromuscular junction of normal and p/q Ca 2+ channel knockout mouse

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming α 1 and the regulatory α 2δ, γ and β subunits. Molecular and functional studies have revealed which α 1 subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by α 1A subunit, mediate evoked transmitter release. The generation of α 1A-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs α 1 and β subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The α 1A subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits α 1B, α 1D and α 1E were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the α 1A. On the other hand, the β 1b, β 2a and β 4 were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the α 1A subunit, P/Q channel under certain pathological conditions.

Effect of d-2 amino-5-phosphonopentanoate and nifedipine on postsynaptic calcium changes associated with long-term potentiation in hippocampal CA1 area

The induction of long-term potentiation (LTP) in CA1 hippocampal area requires a rise in intracellular postsynaptic calcium. Two major calcium mechanisms may mediate the transmembrane calcium influxes that contribute to this calcium accumulation: the N-methyl- d-aspartate (NMDA) receptor channels, which are voltage dependent and have large calcium permeability and voltage-dependent calcium channels (VDCCs). We have addressed the relative contribution of these routes of calcium entry before and during LTP expression, in synaptically evoked dendritic calcium transients from a population of CA1 pyramidal neurons. Combining the use of the fluorescent calcium indicator Fura-2 with field potential measurements, we observed that the calcium transients evoked by single stimuli, during the maintenance phase of LTP, were enhanced. These transients were not affected by d-2 amino-5-phosphonopentanoate ( d-APV) (50 μM), an antagonist of NMDA receptors but were reduced by approximately one-quarter, in the presence of the L-type VDCCs blocker nifedipine (10 μM). During tetanic stimulation (100 Hz, 1 s) the components triggered by the activation of those two calcium mechanisms had comparable magnitudes representing the sum about half of the intracellular calcium accumulation. Thus, following both single and high frequency stimulation, a substantial fraction of calcium entry may occur through other types of VDCCs or be due to calcium release from intracellular stores.

Electrical properties of T-type Ca channels

Neurons possess multiple types of voltage-dependent calcium channels. These channels are classified as either low-voltage-activated (LVA/T-type) or high-voltage-activated (HVA) consisting of L, N, P, Q and R subtypes. T-type Ca channels can be opened by small depolarization that are under the threshold for action potential generation. These T-type Ca channels may be involved in the sub threshold depolarizations that underlies neuronal bursting activities in neurons. Such burst firing might originate in the postsynaptic membrane of the neuronal dendrites.

Calcium channels involved in neurotransmitter release at adult, neonatal and P/Q-type deficient neuromuscular junctions (Review).

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (Ica) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca(2+) inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca(2+)-inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.

Molecular mechanism of exocytosis in neural and immune system

Exocytosis is a common process for the secretion of physiologically active substances such as neurotransmitter, hormone, and inflammatory mediators. Exocytosis is triggered by an increase in intracellular calcium ion concentration. At the nerve terminal, voltage dependent calcium channels (VDCCs) are responsible for this calcium increase. There are several types of VDCC which are different from each other in their electro-physiological and pharmacological characteristics. In order to identify the types of VDCC at the cholinergic nerve terminal, acetylcholine (ACh) release from the electric organ synaptosomes was measured in the presence of type-specific channel blockers. At least three types of VDCC were involved in the ACh release, and N- and P/Q-type VDCC had a major contribution. Adenosine receptor A1 was coupled with N-type VDCC and had negative feedback regulation of ACh release, while A2 receptor coupled with P/Q-type VDCC enhanced the ACh release. Investigation of the inhibitory effects of antibodies from patients of autoimmune disease Lambert-Eaton syndrome on ACh release revealed that P/Q-type channel was a target for the autoantibodies. Unlike the nerve terminal, little is known about the mechanism and molecules involved in the exocytosis of immune cells. Ion channel activities of secretory granule proteins of mast cells were observed. The calcium dependency of the open probability of the channel was similar to that of histamine release from mast cells. We also showed the expression of some SNARE proteins in RBL-2H3 cells. Localization and dynamics of VAMP-7 and syntaxin-3 after antigen stimulation suggested the involvement of SNARE proteins in the exocytosis of mast cells.

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; dl 50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, ω-conotoxin-GVIA and ω-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 μM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker ω-conotoxin-GVIA (1 μM) inhibited neurotransmitter release by 50–70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker ω-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and ω-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.

L-type calcium channels unmasked by cell-permeant Ca2+ buffer at mouse motor nerve terminals.

The involvement of the different types of voltage-dependent calcium channels (VDCC) in both DM-BAPTA-AM-incubated and EGTA-AM-incubated mature mice levator auris neuromuscular junctions (NMJ) was studied. We evaluated the effects of omega-agatoxin IVA (omega-Aga IVA), nitrendipine and omega-conotoxin GVIA (omega-CgTX) (P/Q-, L- and N-type VDCC blockers, respectively) on perineurial calcium currents (ICa) and nerve-evoked transmitter release. The application of omega-Aga IVA (100 nM) drastically reduced perineurial ICa (>90%) and nerve-evoked transmitter release (>90% of reduction in quantal content, m) at both DM-BAPTA-AM-incubated and EGTA-AM-incubated NMJ. The L-type VDCC antagonist nitrendipine (10 microM) caused a significant reduction (23+/-9%, n=5) of perineurial ICa at DM-BAPTA-AM-incubated NMJ. In addition, after the block of P/Q-type VDCC with omega-Aga IVA (100 nM), nitrendipine reduced (>90%, n=2) the remaining perineurial ICa. Such reduction was not observed at EGTA-AM-incubated NMJ, before or after the total block of P/Q-type VDCC. Moreover, nitrendipine did not significantly reduce the quantal content of DM-BAPTA-AM-incubated NMJ. Finally, the application of omega-CgTX (5 microM) did not significantly affect perineurial ICa or nerve-evoked transmitter release at either DM-BAPTA-AM-incubated or EGTA-AM-incubated NMJ. These results show the existence of a nitrendipine-sensitive, L-type component of perineurial ICa in DM-BAPTA-AM-incubated NMJ of mature mice.

Gangliosides and sialylcholesterol as modulators of synaptic functions.

Gangliosides were shown to enhance the release of acetylcholine from synaptosomes on stimulation. The influx of calcium ion into synaptosomes on membrane depolarization was increased by gangliosides. This was hypothesized to be an underlying mechanisms for the enhancement of acetylcholine release. Studies using calcium channel blockers revealed that four distinct types of voltage-dependent calcium channels occurred in cerebrocortical synapses, and that the N-type was primarily responsible for the evoked release of acetylcholine. An additional result suggests that gangliosides may act mainly on the N-type calcium channel. Cholinergic-specific gangliosides, Chol-1 alpha, were assumed to participate in the mechanism of high-affinity choline uptake. These two different actions of gangliosides were found to be mimicked by synthetic ganglioside analogs. Calcium influx was increased by alpha-sialylcholesterol, and choline uptake was accelerated by beta-sialylcholesterol. Gangliosides and sialylcholesterol having these apparently beneficial effects were shown to ameliorate decreased functions of synapses from aged brains.

Lambert-Eaton syndrome antibodies inhibit acetylcholine release and P/Q-type Ca2+ channels in electric ray nerve endings.

1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.

Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J.

In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > omega-agatoxin IVA > omega-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist omega-conotoxin GVIA was without effect. The P-type channel antagonist omega-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P, N) with the L-type predominantly involved in depolarization induced amylase secretion.

Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction.

1. The involvement of the different types of voltage-dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L-, P/Q- and N-type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve-evoked transmitter release was blocked by omega-agatoxin IVA (omega-AgaIVA), a P/Q-type VDCC blocker, both in normal and reinnervating endplates (100 nM omega-AgaIVA caused > 90% inhibition). The N-type VDCC antagonist omega-conotoxin GVIA (omega-CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1-10 microM), an L-type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40-69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ entry through either P- or L-type VDCCs. Neither 100 nM omega-AgaIVA nor 10 microM nitrendipine affected the miniature endplate potential (MEPP) frequency or amplitude. 4. At the newly formed endplates, K(+)-evoked release was dependent on Ca2+ entry through VDCCs of the P-type family (100 nM omega-AgaIVA reduced approximately 70% of the K(+)-evoked MEPP frequency). L-type VDCCs were found not to participate in this type of release (10 microM nitrendipine lacked any effect). 5. In reinnervating muscles, the L-type VDCC blocker, nitrendipine (10 microM), provoked a significant increase (approximately 25%) in the latency of the evoked endplate potential (EPP). This drug also caused an increase (approximately 0.3 ms) in the latency of the presynaptic currents. The P/Q- and Ny-type VDCC blockers did not affect the latency of the EPP. 6. These results show that at newly formed mouse neuromuscular junctions, as occurs in mature preparations, VDCCs of the P-type family play a prominent role in evoked transmitter release whereas N-type channels are not involved in this process. In addition, signal conduction and transmitter release become highly sensitive to nitrendipine during reinnervation. This suggests that L-type VDCCs may play a role in synaptic transmission at the immature mammalian neuromuscular junction.

Involvement of P-type calcium channels in high potassium-elicited release of neurotransmitters from rat brain slices

Several types of voltage-dependent calcium channels appear to occur in neurons, although coupling of the particular subtype of calcium channels to the release of neurotransmitter has not been clearly understood. We have examined the effects of subtype-specific inhibitors of the calcium channels on depolarization-induced release of endogenous neurotransmitters from brain slices. High potassium-induced release of glutamate and aspartate from hippocampal and striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from the hippocampal slices with almost the same potency as in the case of glutamate, whereas the potency in blocking the release of serotonin and dopamine from striatal slices was lower than that from the hippocampal slices. Another calcium channel blocker, omega-agatoxin TK, that was recently found to block P-type channels with very similar selectivity and potency to omega-agatoxin IVA, also inhibited the release of amino acid transmitters and monoamines, though its potency was lower than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA, partially inhibited the neurotransmitter release, but an L-type channel blocker, nifedipine was ineffective. We propose that the activation of P-type calcium channels makes a major contribution to depolarization-elicited neurotransmitter release in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and omega-agatoxin TK modulate the neurotransmitter release.

Three novel types of voltage-dependent calcium channels in rat cerebellar neurons

With the aim of characterizing the functional and pharmacological properties of the different voltage-dependent Ca2+ channels expressed in a given type of CNS neuron, we obtained single Ca2+ channel recordings from rat cerebellar granule cells in primary culture. Our data show that three novel classes of voltage-dependent Ca2+ channels are coexpressed in cerebellar granule cells. They are pharmacologically distinct from dihydropyridine-sensitive L-type and omega-conotoxin-sensitive N-type channels, and their functional properties are different from those of P- and T-type channels. The three novel 21 pS G1-, 15 pS G2-, and 20 pS G3-type Ca2+ channels have similar inactivation properties. They show complete steady-state inactivation at -40 mV and their single-channel average currents have both sustained and decaying components. They differ in activation threshold (-40 mV for G2, -30 mV for G3, and -10 mV for G1, with 90 mM Ba2+ as charge carrier), mean open time (1.2 msec for G2, 1 msec for G3, 0.8 msec for G1), and single-channel currents (at 0 mV: 0.5 pA for G2, 0.8 pA for G3, and 1.4 pA for G1). Together with the previously characterized multiple L-type Ca2+ channels (Forti and Pietrobon, 1993), G1-, G2-, and G3-type channels constitute the large majority of Ca2+ channels of cerebellar granule cells in culture. The low activation threshold of G2-type channels and their inactivation properties suggest that they might be native counterparts of the recently expressed rat brain clone rbE-II (Soong et al., 1993).