Ni2+ Slows the Activation Kinetics of High-Voltage-Activated Ca2+ Currents in Cortical Neurons: Evidence for a Mechanism of Action Independent of Channel-Pore Block

The effects of Ni²⁺ were evaluated on slowly-decaying, high-voltage-activated (HVA) Ca²⁺ currents expressed by pyramidal neurons acutely dissociated from guinea-pig piriform cortex. Whole-cell, patch-clamp recordings were performed with Ba²⁺ as the charge carrier. Ni²⁺ blocked HVA Ba²⁺ currents (I _Bas) with an EC₅₀ of approximately 60 μm. Additionally, after application of nonsaturating Ni²⁺ concentrations, residual currents activated with substantially slower kinetics than both total and Ni²⁺-sensitive I _Bas. None of the pharmacological components of slowly decaying, HVA currents activated with kinetics significantly different from that of total currents, indicating that the effect of Ni²⁺ on I _Bas kinetics cannot be attributed to the preferential inhibition of a fast-activating component. The effect of Ni²⁺ on I _Ba amplitude was voltage-independent over the potential range normally explored in our experiments (−60 to +20 mV), hence the Ni²⁺-dependent decrease of I _Ba activation rate is not due to a voltage- and time-dependent relief from block. Moreover, Ni²⁺ significantly reduced I _Ba deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The dependence on Ni²⁺ concentration of the I _Ba activation-rate reduction was remarkably different from that found for I _Ba block, with an EC₅₀ of ∼20 μm and a Hill coefficient of ∼1.73 vs.∼1.10. These results demonstrate that Ni²⁺, besides inhibiting the I _Bas under study probably by exerting a blocking action on the pore of the underlying Ca²⁺ channels, also interferes with Ca²⁺-channel gating kinetics, and strongly suggest that the two effects depend on Ni²⁺ occupancy of binding sites at least partly distinct. Received: 13 July 2000/Revised: 9 November 2000     

G-Protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca²⁺ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca²⁺ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (50%) in I _Ba amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca²⁺ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Low voltage-activated Ca2+ conductances in thalamic neurons

Low voltage-activated (LVA) Ca²⁺ conductances were characterized in the neurons of the associative laterodorsal (LD) thalamic nucleus in rat brain slices and in enzymatically isolated thalamic units using electrophysiological techniques. Voltage dependence, kinetics of inactivation, pharmacology, and selectivity of the LVA current in the thalamic neurons from animals older than 14 postnatal days were consistent with the existence of two, “fast” and “slow,” subtypes of LVA Ca²⁺ channels. “Slow” LVA current in enzymatically isolated thalamic neurons was much less prominent, compared with that in slice neurons, suggesting that respective channels are predominatly located on the distal dendrites. “Fast” Ca²⁺ channels were sensitive to nifedipine (K _d−2.6 μM) and La³⁺ (K _d−1.0 mM), whereas “slow” Ca²⁺ channels were sensitive to Ni²⁺ (25 μM). Selectivity of the “fast” Ca²⁺ channels was similar to that found for the LVA Ca²⁺ channels in other preparations (I _Ca:I _Sr:I _Ba−1.0: 1.23: 0.94), while selectivity of the “slow” Ca²⁺ channels more resembled selectivity of the HVA Ca²⁺ channels (I _Ca:I _Sr:I _Ba−1.0: 2.5: 3.4).     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

Background

Dimebon is an antihistamine compound with a long history of clinical use in Russia. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders. It has demonstrated efficacy in phase II Alzheimer's disease (AD) and Huntington's disease (HD) clinical trials. The mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain unclear. It has been suggested that Dimebon may act by blocking NMDA receptors or voltage-gated Ca²⁺ channels and by preventing mitochondrial permeability pore transition.

Results

We evaluated the effects of Dimebon in experiments with primary striatal neuronal cultures (MSN) from wild type (WT) mice and YAC128 HD transgenic mice. We found that Dimebon acts as an inhibitor of NMDA receptors (IC50 = 10 μM) and voltage-gated calcium channels (IC50 = 50 μM) in WT and YAC128 MSN. We further found that application of 50 μM Dimebon stabilized glutamate-induced Ca²⁺ signals in YAC128 MSN and protected cultured YAC128 MSN from glutamate-induced apoptosis. Lower concentrations of Dimebon (5 μM and 10 μM) did not stabilize glutamate-induced Ca²⁺ signals and did not exert neuroprotective effects in experiments with YAC128 MSN. Evaluation of Dimebon against a set of biochemical targets indicated that Dimebon inhibits α-Adrenergic receptors (α_1A, α_1B, α_1D, and α_2A), Histamine H₁ and H₂ receptors and Serotonin 5-HT_2c, 5-HT_5A, 5-HT₆ receptors with high affinity. Dimebon also had significant effect on a number of additional receptors.

Conclusion

Our results suggest that Ca²⁺ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca²⁺ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.     

Characterization of voltage operated R-type Ca2+ channels in modulating somatostatin receptor subtype 2- and 3-dependent inhibition of insulin secretion from INS-1 cells

Somatostatin (SST) inhibits Ca²⁺ entry into pancreatic B-cells via voltage-operated Ca²⁺ channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca²⁺ channels (Ca_V2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca²⁺]_i in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca²⁺]_i. Subsequently, these agonists led to a decrease in [Ca²⁺]_i below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca²⁺]_i. Instead, a persistent minor suppression of [Ca²⁺]_i was detected from 25 min. R-type channel blocker SNX-482 altered [Ca²⁺]_i in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca²⁺]_i and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.     

Peculiarities of Selectivity of Three Subtypes of Low-Threshold T-Type Calcium Channels

Despite the progress in studies of the properties and functions of low-threshold calcium channels (LTCCs) [1], the mechanisms of their selectivity and permeability remain unstudied in detail. We performed a comparative analysis of the selectivity of three cloned pore-forming LTCC subunits (α1G, α1H, and α1I) functionally expressed in Xenopus oocytes with respect to bivalent alkaline-earth metal cations (Ba²⁺, Ca²⁺, and Sr²⁺. The relative conductivities (G) of these channels were determined according to the amplitudes of macroscopic currents (I) and potentials of zero currents (E). The currents were recorded after preliminary intracellular injection of a fast calcium buffer, BAPTA, in order to suppress the endogenous calcium-dependent chloride conductivity. Channels formed by α1G subunits demonstrated the following ratios of the amplitudes of macroscopic currents and potentials of zero current: I _Ca:I _Ba:I _Sr = 1.00:0.75:1.12 and E _Ca ≈ E _Ba ≈ E _Sr. For channels that were formed by α1H and α1I subunits, these ratios were as follows: I _Ca:I _Ba:I _Sr = 1.00:1.20:1.17, E _Ca ≈ E _Ba ≈ E _Sr and I _Ca:I _Ba:I _Sr = 1.00:1.48: 1.45, E _Ca ≈ E _Ba ≈ E _Sr respectively. The different macroscopic conductivities and similar potentials of zero current typical of α1G and α1I channels indicate that, probably, various bivalent cations can in a differential manner influence the stochastic parameters of functioning of these channels. At the same time, channels formed by α1H subunits are characterized by more positive potentials of zero current for Ca²⁺. It seems possible that the selectivity of the above channels is determined by mechanisms that mediate the selectivity of most high-threshold calcium channels (more affine binding of Ca²⁺ inside the pore). Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 319–329, July–August, 2005.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Down-regulation of N-type voltage-activated Ca2+ channels by gabapentin

1. Although the cellular and molecular mechanisms of the anticonvulsant action of gabapentin (GBP) remain incompletely described, in vitro studies have shown that GBP binds to the ₂ subunit of the high voltage-activated (HVA) Ca²⁺ channels.2. In this report, we analyzed the effects of GBP on the functional expression of HVA Ca²⁺ channels in the PC12 cell line model system. Negligible inhibition of Ca²⁺ channel activity was observed after acute treatment, but a significant decrease in Ca²⁺ current amplitude was promoted by chronic exposure to GBP.3. Consistent with this, radioligand binding experiments showed a comparable reduction in the total number of membrane HVA N-type channels after GBP treatment.     

The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1

In T-type Ca²⁺ channels, macroscopic I_Ba is usually smaller than I_Ca, but at high Ca²⁺ and Ba²⁺, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using Ca_V3.1 channels studied under similar experimental conditions (TEA_o and K_i). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, i_Div, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γ_max(Ca²⁺) of 9.5 ± 0.4 pS and γ_max(Ba²⁺) of 10.3 ± 0.5 pS. Apparent K_M was lower for Ca²⁺ (2.3 ± 0.7 mM) than for Ba²⁺ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca²⁺. As predicted by γ, macroscopic G_maxCa was larger than G_maxBa at 5 mM (G_maxCa²⁺/Ba:²⁺1.43 ± 0.14) and similar at 60 mM (G_maxCa²⁺/Ba:²⁺1.10 ± 0.02). However, over the range of activation, I_Ca was larger than I_Ba under both conditions. This was a consequence of the fact that V_rev was more negative for I_Ba than for I_Ca, so that the driving force determining I_Ba was smaller than that determining I_Ca over the range of potentials in standard current-voltage relationships.     

cAMP-dependent regulation of Ca2+ channels expressed inXenopus oocytes

Rat forebrain- and heart-derived mRNA were used to express Ca²⁺ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca²⁺ channel currents (I _Ba, 40 mM Ba²⁺ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI _Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI _Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI _Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I _Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI _Ba, suggesting that the expressed Ca²⁺ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca²⁺ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca²⁺ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.     

Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

Stimulation of 5-HT₃ receptors (5-HT₃Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT₃ receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT₃R channel has high permeability to extracellular calcium (Ca²⁺) and upon stimulation allows increased Ca²⁺ influx. We examined the contribution of Ca²⁺/calmodulin-dependent protein kinase IIα (Ca²⁺/CaMKIIα), interaction of 5-HT₃R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca²⁺ levels in brainstem slices and that the selective 5-HT₃R antagonist palonosetron, can abolish the induced Ca²⁺ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca²⁺ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca²⁺-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT₃R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca²⁺ mobilization via extracellular Ca²⁺ influx through 5-HT₃Rs/L-type Ca²⁺ channels, and intracellular Ca²⁺ release via RyRs on ER, initiate Ca²⁺-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT₃R-mediated, 2-Me-5-HT-evoked emesis.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

Background

The aberrant release of the neurotransmitters, glutamate and calcitonin-gene related peptide (CGRP), from trigeminal neurons has been implicated in migraine. The voltage-gated P/Q-type calcium channel has a critical role in controlling neurotransmitter release and has been linked to Familial Hemiplegic Migraine. Therefore, we examined the importance of voltage-dependent calcium channels in controlling release of glutamate and CGRP from trigeminal ganglion neurons isolated from male and female rats and grown in culture. Serotonergic pathways are likely involved in migraine, as triptans, a class of 5-HT₁ receptor agonists, are effective in the treatment of migraine and their effectiveness may be due to inhibiting neurotransmitter release from trigeminal neurons. We also studied the effect of serotonin receptor activation on release of glutamate and CGRP from trigeminal neurons grown in culture.

Results

P/Q-, N- and L-type channels each mediate a significant fraction of potassium-stimulated release of glutamate and CGRP. We determined that 5-HT significantly inhibits potassium-stimulated release of both glutamate and CGRP. Serotonergic inhibition of both CGRP and glutamate release can be blocked by pertussis toxin and NAS-181, a 5-HT_1B/1D antagonist. Stimulated release of CGRP is unaffected by Y-25130, a 5-HT₃ antagonist and SB 200646, a 5-HT_2B/2C antagonist.

Conclusion

These data suggest that release of both glutamate and CGRP from trigeminal neurons is controlled by calcium channels and modulated by 5-HT signaling in a pertussis-toxin dependent manner and probably via 5-HT₁ receptor signaling. This is the first characterization of glutamate release from trigeminal neurons grown in culture.     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995     

Regulation of Ca2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells

AimsThe effects of acute (100 s) hypoxia and/or acidosis on Ca²⁺ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time.Methods and results1) hiPSC-CMs express two cell populations: rapidly-inactivating I_Ca myocytes (τ_i<40 ms, in 4–5 day cultures) and slowly-inactivating I_Ca (τ_i ≥ 40 ms, in 6–8 day cultures). 2) Hypoxia suppressed I_Ca by 10–20% in rapidly- and 40–55% in slowly-inactivating I_Ca cells. 3) Isoproterenol enhanced I_Ca in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba²⁺ was the charge carrier through the calcium channels, implicating Ca²⁺-dependent inactivation in O₂ sensing. 5) Acidosis suppressed I_Ca by ∼35% and ∼25% in rapidly and slowly inactivating I_Ca cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca²⁺-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells.ConclusionsVariability in I_Ca inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca²⁺-signaling in the two-cell types arises from differential Ca²⁺-dependent inactivation of the Ca²⁺-channel caused by proximity of Ca²⁺-release stores to the Ca²⁺ channels.     

Presence of an acidic glycoprotein in the serum of arthritic rats: Modulation by capsaicin and curcumin

Voltage-dependent L-type Ca²⁺ channels form highly selective pores for Ca²⁺ ions in the membranes of excitable cells. We investigated the functional role of negatively charged residues, within or near the selectivity region, in ion permeation of a human cardiac L-type Ca²⁺ channel. Glutamates in each of the four repeats, and an aspartate in repeat IV, were substituted with positively charged lysine. Wild-type and mutant Ca²⁺ channels were expressed in Xenopus oocytes. Block by Ca²⁺ and Mg² of inward Li⁺ currents through the channels was used to assess the effects of amino acid substitutions on high-affinity divalent cation binding. The rank order of IC₅₀'s for Ca²⁺ block of I_Li was: E677K > E1086K > E334K > E1387K > D1391K > wild-type. The order of IC₅₀'s for Mg²⁺ block of I_Li indicated differential involvement of the same residues in Mg²⁺ binding: E1387K > E334K > E1086K > E677K > D1391K wild-type. Mutants E1387K and D1391K effectively permeated Ba²⁺, but exhibited a decreased single-channel conductance. The unitary current amplitude carried by Na+, in the absence of external divalent cations, was slightly decreased in the E1387K mutant but not in the D1391K mutant. The results confirm that each of the four glutamates participate unequally in high-affinity Ca²⁺ binding. Additionally, our results indicate that these glutamate residues participate in Mg²⁺ binding. The glutamate at position 1387 may be only peripherally involved in the formation of a high-affinity Ca²⁺ -binding site but is central to a Mg²⁺ binding site accessible from the external side of the pore. The aspartate at position 1391 is most likely located just external to the selectivity region. (Mol Cell Biochem 166: 125-134, 1997)     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells

Our previous study demonstrated that a large-conductance Ca²⁺-activated K⁺ current (BK_Ca), a voltage-gated TTX-sensitive sodium current (I_Na.TTX), and an inward rectifier K⁺ current (I_Kir) were heterogeneously present in most of human cardiac c-kit⁺ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit⁺ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BK_Ca with paxilline, but not I_Na.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of I_Kir with Ba²⁺ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BK_Ca channels, but not I_Na.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit⁺ progenitor cells.