N-type Channel Blocker Research Articles

High-voltage-activated Ca 2+ currents show similar patterns of expression in stellate and pyramidal cells from rat entorhinal cortex layer II

High-voltage-activated (HVA) Ca 2+ currents were studied in acutely isolated neurons from rat entorhinal cortex (EC) layer II. Stellate and pyramidal cells, the two main neuronal types of this structure, were visually identified based on morphological criteria. HVA currents were recorded by applying the whole-cell, patch-clamp technique, using 5-mM Ba 2+ as the charge carrier. In both neuronal types, the amplitude of total HVA Ba 2+ currents ( I Bas) showed a significant tendency to increase with postnatal age in the time window considered [postnatal day 15 (P15) to P28–29]. At P20–P29, when I Ba expression reached stable levels, I Ba density per unit of membrane area was not different in stellate versus pyramidal cells. The same was also observed when Ca 2+, instead of Ba 2+, was used as the charge carrier. The pharmacological current subtypes composing total HVA currents were characterized using selective blockers. Again, no significant differences were found between stellate and pyramidal cells with respect to the total–current fractions attributable to specific pharmacological Ca 2+ channel subtypes. In both cell types, ∼52–55% of total I Bas was abolished by the L-type channel blocker, nifedipine (10 μM), ∼23–30% by the N-type channel blocker, ω-conotoxin GVIA (1 μM), ∼22–24% by the P/Q-type channel blocker, ω-agatoxin IVA (100 nM), and ∼11–13% remained unblocked (R-type current) after simultaneous application of L-, N-, and P/Q-type channel blockers. The Ca v 2.3 (α 1E) channel blocker, SNX-482 (100 nM), abolished ∼57–62% of total R-type current. We conclude that HVA Ca 2+ currents are expressed according to similar patterns in the somata and proximal dendrites of stellate and pyramidal cells of rat EC layer II.

Limited regulation of somatodendritic dopamine release by voltage‐sensitive Ca2+channels contrasted with strong regulation of axonal dopamine release

The mechanism underlying somatodendritic release of dopamine (DA) appears to differ from that of axon-terminal release. Specifically, somatodendritic DA release in the substantia nigra pars compacta (SNc) persists in low extracellular Ca2+ concentrations that are insufficient to support axonal release in striatum, suggesting that limited Ca2+ entry is necessary to trigger somatodendritic release. Here, we compared the role of voltage-dependent Ca2+ channels in mediating DA release in striatum versus SNc using specific blockers of N-, P/Q-, T-, R- and L-type Ca2+ channels individually and in combination. Release of DA evoked by a single stimulus pulse in the dorsal striatum and SNc of guinea-pig brain slices was monitored in real time using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Single-pulse evoked DA release was shown to be independent of regulation by concurrently released glutamate or GABA acting at ionotropic receptors in both regions. Under these conditions, striatal DA release was completely prevented by an N-type channel blocker, omega-conotoxin GVIA (100 nm), and was decreased by 75% by the P/Q-type channel blocker omega-agatoxin IVA (200 nm). Blockade of T-type channels with Ni2+ (100 microm) or R-type channels with SNX-482 (100 nm) decreased axonal release in striatum by 25%, whereas inhibition of L-type channels with nifedipine (20 microm) had no effect. By contrast, none of these Ca2+-channel blockers altered the amplitude of somatodendritic DA release in the SNc. Even a cocktail of all blockers tested did not alter release-signal amplitude in the SNc, although the duration of the release response was curtailed. The limited involvement of voltage-dependent Ca2+ channels in somatodendritic DA release provides further evidence that minimal Ca2+ entry is required to trigger the release process, compared with that required for axon-terminal release.

L-type calcium channels mediate transmitter release in isolated, wide-field retinal amacrine cells.

Transmitter release in neurons is triggered by intracellular Ca2+ increase via the opening of voltage-gated Ca2+ channels. Here we investigated the voltage-gated Ca2+ channels in wide-field amacrine cells (WFACs) isolated from the white-bass retina that are functionally coupled to transmitter release. We monitored transmitter release through the measurement of the membrane capacitance (Cm). We found that 500-ms long depolarizations of WFACs from -70 mV to 0 mV elicited about a 6% transient increase in the Cm or membrane surface area. This Cm jump could be eliminated either by intracellular perfusion with 10 mM BAPTA or by extracellular application of 4 mM cobalt. WFACs possess N-type and L-type voltage-gated Ca2+ channels. Depolarization-evoked Cm increases were unaffected by the specific N-type channel blocker omega-conotoxin GVIA, but they were markedly reduced by the L-type blocker diltiazem, suggesting a role for the L-type channel in synaptic transmission. Further supporting this notion, in WFACs the synaptic protein syntaxin always colocalized with the pore-forming subunit of the retinal specific L-type channels (Cav1.4 or alpha1F), but never with that of the N-type channels (Cav2.2 or alpha1B ).

Pre- and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons.

Activation of voltage-dependent Ca2+ channels (VDCCs) is critical for neurotransmitter release, neuronal excitability and postsynaptic Ca2+ signalling. Antagonists of VDCCs can be antinociceptive in different animal pain models. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the processing of pain-related information, but the role of VDCCs to the activity-dependent Ca2+ increase in lamina I neurons and to the synaptic transmission between nociceptive afferents and second order neurons in lamina I is not known. This has now been investigated in a lumbar spinal cord slice preparation from young Sprague-Dawley rats. Microfluorometric Ca2+ measurements with fura-2 have been used to analyse the Ca2+ increase in lamina I neurons after depolarization of the cells, resulting in a distinct and transient increase of the cytosolic Ca2+ concentration. This Ca2+ peak was reduced by the T-type channel blocker, Ni2+, by the L-type channel blockers, nifedipine and verapamil, and by the N-type channel blocker, omega-conotoxin GVIA. The P/Q-type channel antagonist, omega-agatoxin TK, had no effect on postsynaptic [Ca2+]i. The NMDA receptor channel blocker D-AP5 reduced the Ca2+ peak, whereas the AMPA receptor channel blocker CNQX had no effect. Postsynaptic currents, monosynaptically evoked by electrical stimulation of the attached dorsal roots with C-fibre and Adelta-fibre intensity, respectively, were reduced by N-type channel blocker omega-conotoxin GVIA and to a much lesser extent, by P/Q-type channel antagonist omega-agatoxin TK, and the L-type channel blockers verapamil, respectively. No difference was found between unidentified neurons and neurons projecting to the periaqueductal grey matter. This is the first quantitative description of the relative contribution of voltage-dependent Ca2+ channels to the synaptic transmission in lamina I of the spinal dorsal horn, which is essential in the processing of pain-related information in the central nervous system.

Ca2+ channels and synaptic transmission at the adult, neonatal, and P/Q-type deficient neuromuscular junction.

Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.

Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats.

Functional analysis of GnRH neurons is limited, although these neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiological analysis of GnRH neurons. To identify GnRH neurons, we tagged the neurons with green fluorescence protein by a transgenic technique. A dispersed culture of GnRH neurons was prepared from the transgenic rats. After overnight culture, a perforated patch clamp was applied to the identified GnRH neurons to analyze the Ca2+ currents. In neonatal GnRH neurons, high voltage-activated Ca2+ currents were clearly observed, but low voltage-activated Ca2+ current was negligible. Nimodipine (L-type channel blocker) and omega-conotoxin GVIA (N-type channel blocker) each attenuated the current by approximately 20%. The R-type channel blocker SNX-482 attenuated the current by approximately 55%. Inhibition by the P/Q-type channel blocker omega-agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca2+ currents were observed. Inhibitions by nifedipine, omega-conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by omega-agatoxin IVA was clearly seen in 40-61% of the GnRH neurons examined. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R-, and T-type channels. Expressions of P/Q- and T-type channels are developmentally regulated.

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic–clonic seizures. IC neurons are known to have Ca 2+ channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca 2+ channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca 2+ channels were activated by depolarizing pulses from a holding potential of −90 mV in 10 mV increments using 5 mM barium (Ba 2+) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca 2+ channel currents that could be blocked by 50 μM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 μM, L-type channel blocker), ω-conotoxin GVIA (1 μM, N-type channel blocker), and ω-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) ω-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca 2+ channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 μM nickel and inactivated with τ of 15–50 ms, was considered as R-type Ca 2+ channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca 2+ channel antibodies showed that IC neurons express Ca 2+ channel α 1A, α 1B, α 1C, α 1D, and α 1E subunits. We conclude that IC neurons express functionally all members of HVA Ca 2+ channels, but only a subset of these neurons appear to have developed functional LVA channels.

Role of L-type Ca2+ channels in transmitter release from mammalian inner hair cells. II. Single-neuron activity.

Previously reported changes in the gross sound-evoked cochlear potentials after intracochlear perfusion of nimodipine suggest that dihydropyridine-sensitive Ca2+ channels (L-type) control the sound-evoked release of transmitter from the inner hair cells of the mammalian cochlea. In the present study, we combined recording of the action potentials of single primary auditory afferent neurons with intracochlear perfusion to further investigate the role of voltage-gated Ca2+ channels at this synapse. Spontaneous action potential firing rates were depressed by the L-type channel blocker nimodipine, but were elevated by S(-) BAY K8644, an L-type channel agonist. Sound-evoked responses of single primary afferents were depressed by nimodipine in a manner that was consistent with a block at the inner hair cell-afferent dendrite synapse. Perfusions with solutions containing the N-type channel blocker conotoxin GVIA did not differ in their effects from control artificial perilymph perfusions. The results extend the conclusions of the earlier study by showing that L-type Ca2+ channels are primarily responsible for controlling both spontaneous and sound-evoked transmitter release from inner hair cells. In addition it was found that afferent neurons with widely different spontaneous firing rates were all sensitive to nimodipine and to BAY K8644, suggesting that the multiple synaptic outputs of each inner hair cell are under the control of only one major type of Ca2+ channel.

The Neuromuscular Junctions of the Slow and the Fast Excitatory Axon in the Closer of the CrabEriphia spinifronsAre Endowed with Different Ca2+Channel Types and Allow Neuron-Specific Modulation of Transmitter Release by Two Neuropeptides

Most crustacean muscle fibers receive double excitatory innervation by functionally different motor neurons termed slow and fast. By using specific omega-toxins we show that the terminals of the slow closer excitor (SCE) and the fast closer excitor (FCE) at a crab muscle are endowed with different sets of presynaptic Ca(2+) channel types. omega-Agatoxin, a blocker of vertebrate P/Q-type channels, reduced the amplitude of EPSCs by decreasing the mean quantal content of transmitter release in both neurons by 70-85%, depending on the concentration. We provide the first evidence that omega-conotoxin-sensitive channels also participate in transmission at crustacean neuromuscular terminals and are colocalized with omega-agatoxin-sensitive channels in an axon-type-specific distribution. omega-Conotoxin, a blocker of vertebrate N-type channels, inhibited release by 20-25% only at FCE, not at SCE endings. Low concentrations of Ni(2+), which block vertebrate R-type channels, inhibited release in endings of the SCE by up to 35%, but had little effects in FCE endings. We found that two neuropeptides, the FMRFamide-like DF(2) and proctolin, which occur in many crustaceans, potentiated evoked transmitter release differentially. Proctolin increased release at SCE and FCE endings, and DF(2) increased release only at FCE endings. Selective blocking of Ca(2+) channels by different omega-toxins in the presence of peptides revealed that the target of proctolin-mediated modulation is the omega-agatoxin-sensitive channel (P/Q-like), that of DF(2) the omega-conotoxin-sensitive channel (N-like). The differential effects of these two peptides allows fine tuning of transmitter release at two functionally different motor neurons innervating the same muscle.

Atrial natriuretic factor (ANF) effects on L-, N-, and P/Q-type voltage-operated calcium channels.

1. We have previously reported that atrial natriuretic factor (ANF) decreases neuronal norepinephrine (NE) release. The mechanism that mediates NE release from presynaptic membrane to synaptic cleft is a strongly calcium-dependent process. The modulator effect of ANF may be related to modifications in calcium influx at the presynaptic nerve ending by interaction with voltage-operated calcium channels (VOCCs). 2. On this basis we investigated the effects of ANF on K+-induced 45Ca2+ uptake and evoked neuronal NE release in the presence of specific L-, N-, and P/Q-type calcium channel blockers in the rat hypothalamus. 3. Results showed that ANF inhibited K+-induced 45Ca2+ uptake in a concentration-dependent fashion. Concentration-response curves to VOCC blockers nifedipine (NFD, L-type channel blocker), omega-conotoxin GVIA (CTX, N-type channel blocker), and omega-agatoxin IVA (AGA, P/Q-type channel blocker) showed that all the blockers decreased NE release. Incubation of ANF plus NFD showed an additive effect as compared to NFD or ANF alone. However, when the hypothalamic tissue was incubated in the presence of ANF plus CTX or AGA there were no differences in neuronal NE release as compared to calcium channel blockers or ANF alone. 4. These results suggest that ANF decreases NE release by an L-type calcium channel independent mechanism by inhibiting N- and/or P/Q-type calcium channels at the neuronal presynaptic level. Thus, ANF modulates neuronal NE release through different mechanisms involving presynaptic calcium channel inhibition.

Decreased calcium influx into the neonatal rat motor nerve terminals can recruit additional neuromuscular junctions during the synapse elimination period

Individual skeletal muscle fibers in newborn vertebrates are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in an activity-dependent process of synaptic elimination by axonal competition. Because synaptic activity depends strongly on the influx of calcium from the external media via presynaptic voltage-dependent calcium channels, we investigate the relationship between calcium channels, synaptic activity and developmental axonal elimination. We studied how several calcium channel blockers affect (after 1 h of incubation) the total number of functional axons per muscle fiber (poly-innervation index) of the Levator auris longus muscle of 6-day-old rats. We determined the poly-innervation index by gradually raising the stimulus amplitude and recorded the recruitment of one or more axons that produced a stepwise increment of the endplate potential. The L-type channel blocker nitrendipine (1 μM) increased the mean poly-innervation index (35.79%±3.91; P<0.05). This effect was not washed out with normal Ringer, although the poly-innervation index returned to the control value when high-calcium Ringer (5 mM) was used. The P-type channel blocker ω-agatoxin-IVA (100 nM) also increased the number of recruitable endplate potentials (27.49%±1.78; P<0.05), whereas N-type channel blocker ω-conotoxin-GVIA (1 μM) was ineffective ( P>0.05). However, neither nitrendipine nor ω-agatoxin-IVA modified the poly-innervation index on high-calcium Ringer ( P>0.05 in both cases). A more intense inhibition of calcium influx (by the sequential use of two calcium channel blockers) did not recruit any additional silent synapses. Moderately increasing the magnesium ions (by 500 μM) in the physiological solution produces a synaptic recruitment (36.78%±2.1; P<0.05) similar to that with L- and P-type calcium channel blockers incubation. This magnesium effect was not washed with normal Ringer but a Ringer that is high in calcium can reverse it. The recruited endings were identified by selective activity-dependent loading with styryl dyes. Rhodaminated α-bungarotoxin-labeled acetylcholine receptors were present in the postsynaptic counterpart. Based on these findings we suggest that, before their complete retraction, functionally silent nerve terminals can be manifested or recovered if calcium influx is reduced by a calcium channel blocker or if external magnesium is increased. The normal activation of this calcium-dependent silencing mechanism during development may be related to the final loss of the supernumerary axons.

Gastrin and the neuropeptide PACAP evoke secretion from rat stomach histamine-containing (ECL) cells by stimulating influx of Ca2+ through different Ca2+ channels.

1. Gastrin and PACAP stimulate secretion of histamine and pancreastatin from isolated rat stomach ECL cells. We have examined whether or not secretion depends on the free cytosolic Ca2+ concentration ([Ca2+]i) and the pathways by which gastrin and PACAP elevate [Ca2+]i. Secretion was monitored by radioimmunoassay of pancreastatin and changes in [Ca2+]i by video imaging. The patch clamp technique was used to record whole-cell currents and membrane capacitance (reflecting exocytosis). 2. In the presence of 2 mM extracellular Ca2+, gastrin and PACAP induced secretion and raised [Ca2+]i. Without extracellular Ca2+ (or in the presence of La3+) no secretion occurred. The extracellular Ca2+ concentration required to stimulate secretion was 10 times higher for gastrin than for PACAP. Depletion of intracellular Ca2+ pools by thapsigargin had no effect on the capacity of gastrin and PACAP to stimulate secretion. 3. Gastrin-evoked secretion was inhibited 60-80 % by L-type channel blockers and 40 % by the N-type channel blocker omega-conotoxin GVIA. Combining L-type and N-type channel blockers did not result in greater inhibition than L-type channel blockers alone. Whole-cell patch clamp measurements confirmed that the ECL cells are equipped with voltage-dependent inward Ca2+ currents. A 500 ms depolarising pulse from -60 mV to +10 mV which maximally opened these channels resulted in an increase in membrane capacitance of 100 fF reflecting exocytosis of secretory vesicles. 4. PACAP-evoked secretion was reduced 40 % by L-type channel blockers but was not influenced by inhibition of N-type channels. SKF 96365, a blocker of both L-type and receptor-operated Ca2+ channels, inhibited PACAP-evoked secretion by 85 %. Combining L-type channel blockade with SKF 96365 abolished PACAP-evoked secretion. 5. The results indicate that gastrin- and PACAP-evoked secretion depends on Ca2+ entry and not on mobilisation of intracellular Ca2+. While gastrin stimulates secretion via voltage-dependent L-type and N-type Ca2+ channels, PACAP acts via L-type and receptor-operated Ca2+ channels.

The combined effects of N-type calcium channel blockers and morphine on A delta versus C fiber mediated nociception.

Intrathecal mu opiates produce analgesia presynaptically by inhibiting calcium ion influx and postsynaptically by increasing potassium flux. Mu receptors are expressed on presynaptic terminals of unmyelinated (C), but not myelinated (A delta) nociceptors. Thus, mu-opioids such as morphine may act presynaptically to inhibit C, but not A delta, neurotransmission, and postsynaptically on dorsal horn cells that receive input from A delta and/or C fiber nociceptors. N-type calcium ion channel blockers, such as omega-conotoxin GVIA (omega-CTX), produce analgesia by impeding flux of calcium ions into A delta and C fiber nociceptor terminals. Thus, morphine and omega-CTX attenuated C fiber nociception additively, possibly indicating the same presynaptic site of action. Conversely, morphine and omega- CTX were supraadditively analgesic on an A delta test, indicating that these agents probably have different sites of action. We conclude that although intrathecal application of either morphine or omega-CTX attenuates both A delta and C fiber mediated nociception in rats, the combined effects are quite different for the two fiber types. Specifically, although coadministration of morphine with omega-CTX produces an additive, apparently presynaptic antinociception for C fiber-mediated responses, the combination produces a clearly supraadditive, and likely synergistic effect on A delta mediated nociception, probably by acting at pre and postsynaptic sites, respectively. This study demonstrates that combined spinal administration of mu opioids and N-type calcium channel blockers may be useful in providing analgesia for A delta mediated (first, sharp) pain while minimizing the side effects of both drugs.

Associative Long-Term Depression in the Hippocampus Is Dependent on Postsynaptic N-Type Ca2+Channels

Long-term depression (LTD) is a form of synaptic plasticity that can be induced either by low-frequency stimulation of presynaptic fibers or in an associative manner by asynchronous pairing of presynaptic and postsynaptic activity. We investigated the induction mechanisms of associative LTD in CA1 pyramidal neurons of the hippocampus using whole-cell patch-clamp recordings and Ca(2+) imaging in acute brain slices. Asynchronous pairing of postsynaptic action potentials with EPSPs evoked with a delay of 20 msec induced a robust, long-lasting depression of the EPSP amplitude to 43%. Unlike LTD induced by low-frequency stimulation, associative LTD was resistant to the application of d-AP-5, indicating that it is independent of NMDA receptors. In contrast, associative LTD was inhibited by (S)-alpha-methyl-4-carboxyphenyl-glycine, indicating the involvement of metabotropic glutamate receptors. Furthermore, associative LTD is dependent on the activation of voltage-gated Ca(2+) channels by postsynaptic action potentials. Both nifedipine, an L-type Ca(2+) channel antagonist, and omega-conotoxin GVIA, a selective N-type channel blocker, abolished the induction of associative LTD. 8-hydroxy-2-dipropylaminotetralin (OH-DPAT), a 5-HT(1A) receptor agonist, inhibited postsynaptic Ca(2+) influx through N-type Ca(2+) channels, without affecting presynaptic transmitter release. OH-DPAT also inhibited the induction of associative LTD, suggesting that the involvement of N-type channels makes synaptic plasticity accessible to modulation by neurotransmitters. Thus, the modulation of N-type Ca(2+) channels provides a gain control for synaptic depression in hippocampal pyramidal neurons.

Different distribution of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat hippocampal neurons.

The distribution patterns of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat primary cultured hippocampal neurons were investigated with a confocal laser-scanning microscope. Cells were loaded with the calcium indicator dye Oregon Green/AM, and the responses to high potassium (90 mM) solution with and without the existence of L- and N-type Ca2+ channel blockers were imaged. In general, extracellular application of high [K+] solution induced a [Ca2+]o-dependent increase of [Ca2+]i in both somata and dendritic processes. The increase was reduced by a N-type channel blocker, omega-conotoxin GVIA, and the reduction was greater in dendritic processes than in somata and proximal dendrites. In contrast, the reduction induced by the L-type calcium channel blocker, nifedipine, was observed evenly all over the neurons. The results demonstrated the heterogeneous distribution of nifedipine- and omega-conotoxin GVIA-sensitive calcium channels in cultured hippocampal neurons.

N-Type calcium channels control sympathetic neurotransmission in human heart atrium.

Because knowledge about the type of calcium channels involved in action potential-induced norepinephrine release from the human peripheral sympathetic nervous system is sparse, we investigated which types of calcium channels are functionally important in the sympathetic nerves of human cardiac tissue. In superfused segments of human right atrial appendages, the type of calcium channels that control [(3)H]norepinephrine release evoked by transmural electrical stimulation was determined. [(3)H]norepinephrine release was almost abolished by 0.2 micromol/L omega-conotoxin GVIA (a selective blocker of N-type channels) but was not modified by 0.1 micromol/L omega-agatoxin IVA (a selective blocker of P- and Q-type channels). Mibefradil (a T-type and N-type calcium channel blocker) at concentrations of 0.3 to 3 micromol/L reduced the evoked tritium overflow in a frequency- and calcium-dependent manner, whereas 0.1 to 10 micromol/L amlodipine, diltiazem, and verapamil (selective blockers of L-type channels) were ineffective. Norepinephrine release from cardiac sympathetic nerves is triggered by Ca(2+) influx via N-type but not L- and P/Q-type calcium channels. The inhibitory effect of mibefradil on norepinephrine release at clinically relevant concentrations is probably due to its blocking action on N-type Ca(2+) channels. This property of mibefradil is unique among the calcium channel blockers that have been or still are therapeutically applied and may considerably contribute to its slight negative chronotropic effect in vivo.

Ca 2+ channels that activate Ca 2+-dependent K + currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.

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.

Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals.

Multiple types of voltage-activated calcium (Ca(2+)) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca(2+) influx through N- and L- type voltage-activated Ca(2+) channels in sympathetic neurons to the depolarization-induced activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine (NE) synthesis, and the depolarization-induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both omega-conotoxin GVIA (1 microM), a specific blocker of N-type channels, and nimodipine (1 microM), a specific blocker of L-type Ca(2+) channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K(+), indicating that Ca(2+) influx through both types of channels contributes to enzyme activation. In contrast, K(+) stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by omega-conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K(+) stimulation of NE release from both ganglia and irises was also blocked completely when omega-conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca(2+) influx through N- and L-type Ca(2+) channels.

Biophysical and pharmacological diversity of high-voltage-activated calcium currents in layer II neurones of guinea-pig piriform cortex.

1. High-voltage-activated calcium currents were studied with the whole-cell, patch-clamp technique in acutely dissociated pyramidal neurones from guinea-pig piriform cortex layer II. Barium ions were used as charge carriers. 2. Barium currents (IBa) displayed a remarkable kinetic diversity in different neurones. The ratio between the current amplitude at the end of the test pulses and the peak amplitude (Re/p) showed two frequency-distribution peaks at approximately 0.4 and 0.8. The index of current activation speed (rise time 10-90 %) directly correlated with the index of current persistence, Re/p. 3. The half-activation potential (V ) of total IBas positively correlated with the Re/p of the corresponding currents. This implied that the high-decay IBas also had a more negative voltage range of activation than the more persistent ones. 4. The L- and N-type channel blockers nifedipine (10 microM) and omega-conotoxin GVIA (omega-CTx GVIA, 0.5-1 microM) additively blocked 20 and 25 % of the total IBa, respectively. The P/Q-type calcium channel blockers omega-agatoxin IVA (100 nM), omega-conotoxin MVIIC (1 microM) and 3.3 funnel toxin (1 microM), had little effect on IBa. 5. The nifedipine- and omega-CTx GVIA-sensitive current had a Re/p > 0.55 and their voltage dependence of activation was of the high-voltage-activated type (V approximately 0 mV). 6. High-, intermediate- and low-decay blocker-resistant currents were observed in different neurones. Their Re/p values highly correlated with those of the corresponding total IBas and with the voltage dependence of activation of the underlying conductances. Exponential fittings of the inactivation phase of blocker-resistant currents returned very fast time constants (lower than 30 ms) for high-decay currents (Re/p < 0.25). The intermediate-decay currents (Re/p approximately 0.55) could not derive from variable combinations of high- and low-decay current components. 7. Our data demonstrate a remarkable variety in voltage-activated calcium currents expressed by piriform cortex neurones, that include currents resistant to high-voltage-activated calcium-channel blockers.