Voltage-activated Calcium Channel Currents Research Articles

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

A novel \u03b1-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Additive inhibitory effects of calcitonin and capsaicin on voltage activated calcium channel currents in nociceptive neurones of rat

Calcitonin, a peptide hormone expressed in C-cells of the thyreoid gland, as well as capsaicin, isolated from chili, both, modify intracellular signalling in nociceptive neurones. The pathways triggered by the activation of either of these receptors results in a modulation of the intracellular calcium ([Ca 2+] i) concentration. While the regulation of [Ca 2+] i depends on many factors, voltage activated calcium channels (VACCs) are a major gate for the calcium entry into neurones. Here we describe the changes of voltage gated calcium channel currents ( I Ca(V)) induced by calcitonin and/or capsaicin. Currents were recorded using adequate solutions and voltage protocols with the whole cell patch-clamp technique. When the channels were opened by a depolarisation to 0 mV, both substances reduce the peak I Ca(V) (calcitonin (10 nM): 29.3 ± 3.9%; capsaicin (0.5 μM): 41.1 ± 7.7%). While the effect of calcitonin was voltage dependent, capsaicin shifted the largest current to the more hyperpolarizing range (peak current from −10 to −20 mV). A subsequent co-application of either of the two substances (with a pre-application of either 3 min or 60 min) results in an additive reduction of the currents, and prevents the capsaicin-induced shift of the current–voltage relation. Therefore, we hypothesize, that the activation of either of the two receptors reduces I Ca(V) by different cellular binding sites of the channel protein triggering channel opening. These findings may be useful to understand cellular mechanisms of pain modulation and might help to find better treatments for neuropathic pain.

Anti-allodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and protein kinase C mediated modulation of Ca2+ channels

Flavonoids are increasingly ingested by the population as chemotherapeutic and anti-inflammatory agents. Myricetin is a naturally occurring flavonoid known for its anti-neoplastic and anti-inflammatory effects. Recently, behavioral studies indicate a potential analgesic effect in animal models of pain. Pilot studies suggest a flavonoid-induced modulation of intracellular protein kinases and interactions with voltage activated calcium channels.The aim of this study was to investigate the analgesic effect of myricetin in a neuropathic pain model (spinal nerve ligation, SNL) in rats. To identify potential mechanisms of action, in vitro whole cell patch-clamp recordings of isolated rat dorsal root ganglia (DRG) neurons were performed to analyze the modulation of voltage activated calcium channel currents (ICa(V)) and the influence of intracellular kinase phosphorylation such as p38 mitogen-activated protein kinase (p38) or protein kinase C (PKC).In vivo, a single injection of myricetin (0.1–10mg/kg i.p.) reduced SNL-induced mechanical allodynia and thermal hyperalgesia lasting for several hours. In vitro, ICa(V) (depolarization from −80 to 0mV) were reduced (10–56%) by low (0.1–5μM) concentrations of myricetin. This decrease was abolished by blockade of PKC (20μM chelerythrine for 30min), but not of p38 (10μM SB203580 for 30min). In contrast, higher (10–100μM) concentrations of myricetin induced an increase of ICa(V) (20–40%), which was blocked by inhibition of p38, but not of PKC.We conclude that myricetin transiently reduces established neuropathic pain behavior. This analgesic effect may be related to its PKC-induced decrease of ICa(V) in DRG neurons.

Analgesic α-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in sensory neurons of α9 nicotinic receptor knockout mice

α-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. We have reported previously that the α9α10 nicotinic acetylcholine receptor (nAChR) selective-conotoxins Vc1.1 and Rg1A potently and selectively inhibit high voltage-activated (HVA) N-type calcium channel currents in dissociated neurons from rat dorsal root ganglia (DRG). Our data indicated that Vc1.1 does not interact directly with N-type Ca2+ channels but inhibits them via GABAB receptor activation. The present study investigated Vc1.1 and Rg1A inhibition of N-type Ca2+ channels currents in DRG neurons of wild-type and α9 knockout (KO) mice to determine if the α9 nAChR was necessary for inhibition of the Ca2+ channel current. Application of Vc1.1 (100 nM) inhibited N-type Ca2+ channel currents to 69.2 ± 3.5% of control in DRG neurons isolated from wild-type mice. In {greater than or equal to >70% of DRG neurons isolated from the α9 KO mice, both Vc1.1 and Rg1A selectively inhibited N-type Ca2+ channel currents with an IC50 of 24.6 nM and 22.4 nM, respectively. The GABAB receptor antagonist CGP55845 (1 μM) antagonized the effect of Vc1.1 and Rg1A on the N-type Ca2+ channels in α9 KO mice. RT-PCR and western blot analysis confirmed the absence of the α9 nAChR in mice carrying a null mutation for the nAChR α9 gene. These results demonstrate that the inhibition of N-type Ca2+ channel channels by Vc1.1 and Rg1A is not mediated by the expression of α9α10 nAChRs in DRG neurons.

Sensitization of voltage activated calcium channel currents for capsaicin in nociceptive neurons by tumor-necrosis-factor-α

It is known that application of tumor-necrosis-factor-α (TNF-α) sensitizes neuronal calcium channels for heat stimuli in rat models of neuropathic pain. This study examines whether TNF-α modulates the capsaicin-induced effects after transient receptor potential vanilloid (TRPV)-1 receptor activation on voltage activated calcium channel currents ( I Ca(V)). TRPV-1 receptors are activated by heat and play an important role in the pathogenesis of thermal hyperalgesia in neuropathic pain syndromes, while voltage activated channels are essential for transmission of neuronal signals. Eliciting I Ca(V) in DRG neurons of rats by a depolarization from the resting potential to 0 mV, TNF-α (100 ng/ml) reduces I Ca(V) by 16.9 ± 2.2%, while capsaicin (0.1 μM) decreases currents by 27 ± 4.3%. Pre-application of TNF-α (100 ng/ml) for 24 h results in a sensitization of I Ca(V) to capsaicin (0.1 μM) with a reduction of 42.8 ± 4.4% mediated by TRPV-1. While L-type (36.6 ± 5.2%) and P/Q-type currents (35.6 ± 4.1%) are also sensitized by TRPV-1 activation, N-type channel currents are most sensitive (74.5 ± 7.3%). The capsaicin-induced shift towards the hyperpolarizing voltage range does not occur when TNF-α is applied. Summarizing, TNF-α sensitizes nociceptive neurons for capsaicin.

SnCl 2 reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats

Stannous dichloride (SnCl 2) occurs in the environment where it has been especially enriched in aquatic ecosystems. Furthermore, it is used in food manufacturing (e.g. for stabilizing soft drinks or as an anti-corrosive substance) and in nuclear medicine where it is employed as a reducing agent for technecium-99m (99mTc) and therefore is applied intravenously to human beings. SnCl 2 is known to have toxic effects on the nervous system which can be related to alterations of intracellular calcium homeostasis ([Ca 2+] i). In this study the whole cell patch clamp technique is used on dorsal root ganglion neurons of 3-week-old “Wistar” rats to evaluate the effects of SnCl 2 on voltage-activated calcium channel currents ( I Ca(V)). I Ca(V) were reduced concentration-dependently by SnCl 2 (1–50 μM). 1 μM SnCl 2 reduced I Ca(V) by 8.1 ± 4.5% (peak current) and 19.2 ± 8.9% (sustained current), whereas 50 μM inhibited I Ca(V) by 50.6 ± 4.3% (peak current) and 55.6 ± 11.3% (sustained current). Sustained currents were slightly but not significantly more reduced than peak currents. The effect appeared not to be reversible. The threshold concentration was below 1 μM. The current–voltage relation did not shift which is an indication that different calcium channel subtypes were equally affected. There was a slight but not significant shift of the activation/inactivation curves towards the depolarizing direction. We conclude that voltage-gated calcium channels are affected by Sn 2+ similarly to other divalent metal cations (e.g. Pb 2+ or Zn 2+). The reduction of I Ca(V) could be related to the neurotoxic effects of SnCl 2.

Cisplatin modulates voltage gated channel currents of dorsal root ganglion neurons of rats

The anticancer drug cis-diammindichloroplatin (CDDP, cisplatin) causes severe side effects like peripheral sensitive neuropathy. The toxicity of CDDP has been linked to changes in intracellular calcium homeostasis ([Ca 2+] i ). Voltage activated calcium channel currents ( I Ca(V)) are important for the regulation of [Ca 2+] i ; therefore, this study was designed to examine the effect of CDDP on I Ca(V) in comparison to voltage activated potassium ( I K(V)) and sodium ( I Na(V)) channel currents using the whole cell patch clamp method on dorsal root ganglion neurons of rats. In small neurons (≤Ø20 μm) CDDP reduced peak and sustained I Ca(V) concentration dependently (1–100 μM). The IC 50 was 23.9 ± 4.5 μM (±S.D.) for the peak current with a Hill-coefficient of 0.6 ± 0.1 and 38.8 ± 6.1 μM for the sustained current (Hill-coefficient: 0.7 ± 0.1). I K(V) were reduced by 20.9 ± 4.8% (10 μM) and I Na(V) were only reduced by 9.2% ± 7.2% (10 μM). I Ca(V) of large neurons (≥Ø25 μm) were less sensitive to CDDP. The peak I Ca(V) was reduced by 14.1 ± 2.3% and I K(V) by 12.8 ± 3.4% (100 μM). The sensitivity of I Na(V) in large neurons to CDDP was not different compared to small neurons. We conclude that the reduction of I Ca(V) in small cells may be responsible for the neurotoxic side effects CDDP causes in sensory neurons.

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents ( I Ca(V)). I Ca(V) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba 2+ as charge carrier. In large diameter neurones (>35μm), a concentration of 50μM was needed to reduce I Ca(V) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (≤30μm), the IC 50 was 0.36 μM. This effect was concentration dependent with a threshold below 0.025 μM and maximal blockade (>80%) at 5μM. The current–voltage relation was shifted to the hyperpolarized direction with an increase of the current between −40 and −10mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 μM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I Ca(V) at negative and decreases I Ca(V) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.

Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse.

1. The effects of nociceptin/orphanin FQ (N/OFQ) and opioid receptor agonists on voltage-activated calcium channel currents (I(Ca)) were examined in acutely isolated mouse trigeminal ganglion neurons using whole-cell patch-clamp recordings. These effects were correlated with responses of the neurons to capsaicin and binding of Bandeiraea simplicifolia isolectin B4 (IB4). 2. Trigeminal neurons were divided into two populations based on the presence (type 2) or absence (type 1) of a prominent T-type I(Ca). N/OFQ potently (EC(50) of 19 nM) inhibited high-voltage-activated (HVA) I(Ca) in most (82 %) small (capacitance < 12 pF) type 1 neurons, but few (9 %) larger (> 12 pF) type 1 neurons. N/OFQ inhibited I(Ca) in few (23 %) type 2 cells, and did not affect the T-type I(Ca) in any cell. 3. The mu-opioid agonists DAMGO and morphine inhibited I(Ca) in most type 1 neurons, more often (95 % versus 77 %) in the small cells. The inhibition of I(Ca) by DAMGO and morphine was more efficacious in small versus large type 1 neurons. mu-Opioids did not inhibit I(Ca) in type 2 neurons. 4. Most small type 1 neurons were sensitive to capsaicin (93 %) and bound IB4 (86 %). Fewer larger type 1 neurons responded to capsaicin (30 %) or bound IB4 (58 %). Type 2 neurons did not respond to capsaicin, although some bound IB4 (35 %). 5. Thus, N/OFQ preferentially inhibits HVA I(Ca) in a subpopulation of small nociceptive trigeminal ganglion neurons that is also highly sensitive to mu-opioid agonists.

Developmental regulation of T-, N- and L-type calcium currents in mouse embryonic sensory neurones.

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and omega-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17-18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a -40 mV pulse from a holding potential of -100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17-18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17-18. The density of the N-type current studied by the application of omega-conotoxin-GVIA (1 microM) increased significantly between E13 and E15/E17-18. The use of the dihydropyridine nitrendipine (1 microM) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 microM) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.

Voltage-activated calcium channel currents of rat dorsal root ganglion cells are reduced by trimethyl lead

Using the conventional whole-cell patch-clamp recording technique with cultured neurones of rat dorsal root ganglions (DRG), we analysed the effects of trimethyl lead (TML) on voltage-activated calcium channel currents. TML reduces voltage-activated calcium channel currents in a dose-dependent manner, with a threshold concentration below 0.5 μM and a total reduction of the current (≥80% of the control current) at concentrations above 50 μM. Half of the current is abolished at TML concentrations between 1 and 5 μM. The action is irreversible and is not voltage dependent. After application of TML the current decreases with each activation of the channel until a steady state is reached after 8–12 min, when the channel was activated every 10 s. The channel had to be in the open state for TML to act. TML is a potent compound for reducing voltage activated calcium channel currents. These effects of TML must be taken into account in explaining the neurotoxic effects of this organic metal compound.

Methyl mercury reduces voltage-activated currents of rat dorsal root ganglion neurons.

Methyl mercury (MeHg) is a widespread toxicant with major actions on the nervous system. Since the function of neurons depends on voltage gated ion channels, we examined the effects of micromolar concentrations of methyl mercury on voltage-activated calcium, potassium and sodium channel currents of cultured rat dorsal root ganglion (DRG) neurons. The cells, which were obtained from 2-4 day old rat pups, were whole-cell patch-clamped. Currents were separated by selective intra- and extracellular solutions as well as specific depolarizing voltage steps. We did not distinguish between different calcium, potassium or sodium channel subtypes. All three types of voltage-activated currents were irreversibly reduced by MeHg in a concentration dependent manner. Voltage-activated calcium and potassium channel currents were more sensitive to MeHg (Calcium: IC50 = 2.6 +/- 0.4 microM; Potassium: IC50 = 2.2 +/- 0.3 microM) than voltage-activated sodium channels (IC50 = 12.3 +/- 2.0 microM). The Hill coefficients for the reduction of the currents were calculated as approximately 1 for calcium and potassium channel currents and as 1.7 for sodium currents. In the cases of the voltage-activated calcium and sodium channel currents the reduction was clearly use dependent. Higher concentrations of MeHg (> or = 5 microM) resulted in a biphasic change in the holding membrane current at the potential of -80 mV in approximately 25% of the cases.

Serotonin modulates N- and P-type calcium currents in neocortical pyramidal neurons via a membrane-delimited pathway.

1. The effects of serotonin (5HT) on neocortical pyramidal neurons were studied using whole cell and ON-cell patch-clamp recordings from acutely dissociated neurons. 2. 5HT decreased high voltage-activated calcium channel currents in a dose-dependent and reversible manner in acutely dissociated neocortical pyramidal neurons. The maximum block was 30% of the peak whole cell current (at -10 mV). 3. The 5HT modulation was mimicked by 5HT1A agonists and was reduced by 5HT1A antagonists. 5HT2 antagonists had no effect on the modulation. These data suggest that the 5HT effects were mediated by 5HT1A receptors. 4. The 5HT1A modulation was reduced in the presence of the specific N-type blocker omega-conotoxin GVIA (CgTx) and by the P-type channel blocker omega-agatoxin IVA (AgTx), but not by the L-type blocker nifedipine. 5HT did not modulate the slowed tail currents in the presence of the dihydropyridine agonist Bay K 8644. These data suggest that N- and P-type channels (but not L-type channels) were targeted by 5HT. 5. The modulation involved G proteins and utilized a membrane-delimited pathway. The modulation was rapid in onset (tau approximately 600 ms) and offset. About 50% of the reduction in current by 5HT1A agonists was overcome by prepulses to 120 mV. 6. Slowing of current onset kinetics in response to 5HT1A agonists was seen rarely in neocortical pyramidal neurons (11% of cases). The presence of slowing depended on agonist concentration, being evident only with high micromolar doses.

Synthesis and calcium antagonistic activity of 8-[N-[2-(3,4-dimethoxy- phenyl)ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepi ne fumarate.

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl) ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 microM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 microM, n = 7) and left atria (IC50 = 4 microM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 +/- 5 microM) and low (IC50: 129 +/- 34 microM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 +/- 13 microM) in chick dorsal root ganglion neurons. In addition nicotine-induced currents were potently inhibited (IC50: 6 +/- 0.7 microM) in bovine chromaffin cells.

Calcium channels as target sites of heavy metals

Zinc (Zn), aluminium (Al), mercury (Hg), methylmercury (MeHg) and lead (Pb) extracellulary applied reduce voltage-activated calcium channel currents (VACCCs); Pb and Al also reduce AT-methyl-D-aspartate (NMDA)activated channel currents (NACCs). Pb is most effective in reducing VACCCs, with an IC 50of 0.46 μM, followed by Hg (IC 50 = 1.1 μM) and MeHg (IC 50 = 2.6 μM). Zn and Al were less potent (IC 50 = 69 and 84 μM, respectively). Al acts on channels in the open state; its effect is pH dependent. The effects of Pb were specific for VACCCs and NACCs. Hg, Al and Zn had only minor effects on voltage-activated potassium and sodium channels, while MeHg reduced potassium channel currents ( IC 50 = 2.2 μM) and, at higher concentrations, sodium channel currents ( IC 50 = 12.3 μM). Al also reduced other receptor-activated channel currents. These results demonstrate that a variety of metal species produce different actions at the level of the cell membrane.

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

1. We examined the actions of mercury (Hg2+) and zinc (Zn2+) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique. 2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of -80 to 0 mV (mainly L- and N-currents) were reduced by Hg2+ and Zn2+. The threshold concentration for Hg2+ effects was 0.1 microM and that for Zn2+ was 10 microM. Voltage-activated calcium channel currents were abolished (> 80%) with 5 microM Hg2+ or 200 microM Zn2+. The peak calcium current was reduced to 50% (IC50) by 1.1 microM Hg2+ or 69 microM Zn2+. While Zn2+ was much more effective in reducing the T-type calcium channel current--activated by jumping from -80 to -35 mV--Hg2+ showed some increased effectiveness in reducing this current. 3. The effects of both cations occurred rapidly and a steady state was reached within 1-3 min. While the action of Zn2+ was not dependent on an open channel state, Hg2+ effects depended partially on channel activation. 4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg2+ and with higher concentrations (> 100 microM) of Zn2+. 5. As Zn2+ in the concentration range used had no influence on resting membrane currents, Hg2+ caused a clear inward current at concentrations > or 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of aluminum on voltage-activated calcium channel currents

1. Extracellular and intracellular effects of aluminum (Al) on voltage-activated calcium channel currents (VACCCs) of cultured rat dorsal root ganglion (DRG) neurons were investigated. Al (0.54 to 5.4 micrograms/ml = 20-200 microM) applied extracellularly reduces VACCCs in a concentration-dependent manner. The IC50 was calculated to be 2.3 micrograms/ml (83 microM). All types of VACCCs were similarly reduced by Al treatment. A slight shift of the current-voltage relation to depolarized potentials was observed for higher Al concentrations (> 2 micrograms/ml). The action of Al was found to be use dependent, with little recovery (max. 20%) after wash. 2. The effect of Al was highly pH dependent in the investigated range (pH 6.4 to 7.8). We observed a rightward shift of the concentration-response curve at pH 7.7 (IC50:3.1 micrograms/ml) and a leftward shift at pH 6.4 (IC50:0.56 microgram/ml) compared to the concentration-response curve at pH 7.3. 3. The VACCC declined when 2.7 micrograms/ml Al was added to the internal solution. A steady state was reached within a few minutes. Additional extracellular application of the same concentration lead to an additional decrease of the current. These observations strongly suggest the existence of both intracellular and extracellular accessible binding sites for Al on voltage-activated calcium channels (VACCs). 4. The special characteristics of the action of Al on VACCCs, i.e., the irreversibility, use dependence, and pH dependence, as well as the additional internal binding site may contribute to its neurotoxicity.

Pb2+ reduces voltage- andN-methyl-d-aspartate (NMDA)-activated calcium channel currents

1. While intracellular calcium concentrations are closely regulated, two types of ion channels in neurons allow calcium influx: both voltage-activated and NMDA-activated channels are significantly permeable to calcium. In this study we compare the effects of lead (Pb2+) on currents carried through voltage-activated calcium channels and NMDA-activated channels. 2. Pb2+ reduces voltage-activated calcium channel currents elicited by a voltage jump from -80 to 0 mV at 0.1 to 1 microM, with an IC50 of 0.64 microM and a Hill slope of 1.22. This effect was partially reversible and not voltage dependent. Sodium and potassium currents were relatively unaffected at Pb2+ concentrations sufficient to block calcium channel currents by more than 80%. Pb2+ is, thus, a potent, reversible and selective blocker of voltage-dependent calcium channel currents. 3. A fast reversible and slow irreversible blocking action of Pb2+ was found on NMDA-activated currents. When Pb2+ was applied simultaneously with aspartate and glycine (Asp/Gly), the inward currents were rapidly and reversibly reduced in a dose-dependent manner with a minimum effective concentration below 2 microM and a total blockade (> 80%) with 100 microM Pb2+. The IC50 was approximately 45 microM and the Hill coefficient 1.1. Preincubation with 50 microM Pb2+ resulted in a greater reduction in the response to Asp/Gly/Pb2+. This effect was reversed within 2 to 5 sec of wash. The lack of voltage dependence suggests that Pb2+ does not block the channel but rather alters the binding of agonists. Prolonged superfusion of a cell with the Asp/Gly/Pb(2+)-containing external solution resulted in a slow and irreversible decrease in the Asp/Gly activated current.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian voltage-activated calcium channel currents are blocked by Pb2+, Zn2+, and Al3+.

1. The effects of the di- and trivalent trace metal cations, Pb2+, Zn2+, and Al3+, on voltage-activated calcium channel currents recorded from cultured rat dorsal root ganglion neurons were determined. 2. All three cations blocked transient and sustained components of the voltage-gated calcium channel currents elicited by a voltage jump from -80 mV to 0 mV, but the trace metals differed in threshold, reversibility, and specificity, and in actions on the different components. 3. Pb2+ was most effective in reducing the voltage-activated calcium channel currents. Threshold concentration for Pb2+ was < 0.1 microM. The threshold for Zn2+ action was < 5 microM and that for Al3+ was approximately 20 microM. Total blockade (> 80%) was obtained with concentrations > 1 microM Pb2+, and 150-200 microM Zn2+ or Al3+. Half of the current was blocked with 0.6 microM Pb2+, 69 microM Zn2+, and 84 microM Al3+. The Hill slope for Pb2+ and Zn2+ action was approximately 1, whereas for Al3+ it was close to 3. 4. Al3+ blockade was clearly use dependent, whereas this was not the case for either Pb2+ or Zn2+. 5. The blockade by none of these metals was totally reversible. The best recovery was obtained upon wash after exposure to Pb2+ (> or = 60%), some recovery was seen with Zn2+ (> or = 50%), but there was little or no recovery after application of Al3+. 6. With Zn2+ or Al3+ in the external solution the current-voltage relation often shifted to depolarized voltages.(ABSTRACT TRUNCATED AT 250 WORDS)