High Voltage-activated Ca2 Research Articles

Acute actions of marine toxin latrunculin A on the electrophysiological properties of cultured dorsal root ganglion neurones

The effects of latrunculin A, isolated from the nudibranch Chromodoris sp., on the excitability of neonatal rat cultured dorsal root ganglion neurones were investigated using patch-clamp recording and Ca 2+ imaging techniques. Under current-clamp conditions, acute application of latrunculin A (100 μM) reversibly induced multiple action potential firing and significantly increased action potential duration. No significant effects on action potential peak amplitude, threshold of action potential firing, resting membrane potential and input resistance were observed. Under voltage-clamp conditions, significant and dose-dependent suppression of K + current was seen with 10–100 μM latrunculin A. Additionally, a significant difference between inhibition of the current measured at the peak and the end of a 100 ms voltage step was seen with 100 μM latrunculin A. Fura-2 fluorescence Ca 2+ imaging revealed that latrunculin A (100 μM) significantly inhibited Ca 2+ transients evoked by KCl-induced depolarisation in all neurones. In 36% of DRG neurones, latrunculin A alone had no effect on intracellular Ca 2+. In 64% of neurones, latrunculin A alone evoked a transient rise in intracellular Ca 2+. Moreover, latrunculin A (10–100 μM) significantly inhibited the mean high voltage-activated Ca 2+ current. The effects of latrunculin A on action potential firing and K + currents were attenuated by intracellular phalloidin, an indication that these effects are mediated through actin disruption.

Low Voltage-Activated Ca2+Channels Are Coupled to Ca2+-Induced Ca2+Release in Rat Thalamic Midline Neurons

High voltage-activated Ca2+ channels are coupled to the release of Ca2+ from intracellular stores. Here we present evidence that, in the paraventricular thalamic nucleus and other midline thalamic nuclei, activation of low voltage-activated (LVA) Ca2+ channels stimulates Ca2+-induced Ca2+ release (CICR) from intracellular stores. Voltage-clamp activation of LVA Ca2+ channels in fluo-4 AM-loaded neurons induced an initial transient increase in intracellular Ca2+ concentrations ([Ca2+]i) (mean increase, 19.4%; decay time constant, 71 ms) that reflected the entry of extracellular Ca2+. This was followed by a sustained secondary elevation in [Ca2+]i (mean increase, 4.7%; decay time constant, 7310 ms) that was attributable to CICR. Repeated activation of LVA Ca2+ channels to evoke CICR caused a progressive buildup of baseline [Ca2+]i (mean increase, 13.12 +/- 3.41%) that was reduced by depletion of intracellular Ca2+ stores with thapsigargin or caffeine. In contrast, LVA Ca2+ channel-evoked CICR was absent from ventrolateral thalamocortical relay neurons, suggesting that LVA Ca2+ channel coupling to Ca2+-dependent intracellular signaling may be a property that is unique to nonspecific and midline thalamocortical neurons.

Transfer of β subunit regulation from high to low voltage-gated Ca 2+ channels

High voltage-activated Ca 2+ channel expression and gating is controlled by their β subunits. Although the sites of interaction are known at the atomic level, how β modulates gating remains to be determined. Using a chimeric approach, β subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the α 1 interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where β subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.

Inhibition of High Voltage-Activated Calcium Channels by Spider Toxin PnTx3-6

Animal peptide toxins have become powerful tools to study structure-function relationships and physiological roles of voltage-activated Ca(2+) channels. In the present study, we investigated the effects of PnTx3-6, a neurotoxin purified from the venom of the spider Phoneutria nigriventer on cloned mammalian Ca(2+) channels expressed in human embryonic kidney 293 cells and endogenous Ca(2+) channels in N18 neuroblastoma cells. Whole-cell patch-clamp measurements indicate that PnTx3-6 reversibly inhibited L-(alpha(1C)/Ca(v)1.2), N-(alpha(1B)/Ca(v)2.2), P/Q-(alpha(1A)/Ca(v)2.1), and R-(alpha(1E)/Ca(v)2.3) type channels with varying potency (alpha(1B) > alpha(1E) > alpha(1A) > alpha(1C)) and IC(50) values of 122, 136, 263, and 607 nM, respectively. Inhibition occurred without alteration of the kinetics or the voltage dependence of the exogenously expressed Ca(2+) channels. In N18 cells, PnTx3-6 exhibited highest potency against N-type (conotoxin-GVIA-sensitive) current. In contrast to its effects on high voltage-activated Ca(2+) channels subtypes, application of 1 microM PnTx3-6 did not affect alpha(1G)/Ca(v)3.1 T-type Ca(2+) channels. Based on our study, we suggest that PnTx3-6 acts as a omega-toxin that targets high voltage-activated Ca(2+) channels, with a preference for the Ca(v)2 subfamily (N-, P/Q-, and R-types).

Inhibition of Persistent Sodium Current Fraction and Voltage‐gated L‐type Calcium Current by Propofol in Cortical Neurons: Implications for Its Antiepileptic Activity

Although it is widely used in clinical practice, the mechanisms of action of 2,6-di-isopropylphenol (propofol) are not completely understood. We examined the electrophysiologic effects of propofol on an in vitro model of epileptic activity obtained from a slice preparation. The effects of propofol were tested both on membrane properties and on epileptiform events consisting of long-lasting, paroxysmal depolarization shifts (PDSs) induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine. These results were integrated with a patch-clamp analysis of Na(+) and high-voltage activated (HVA) calcium (Ca(2+)) currents from isolated cortical neurons. In bicuculline, to avoid any interference by gamma-aminobutyric acid (GABA)-A receptors, propofol (3-100 microM) did not cause significant changes in the current-evoked, sodium (Na(+))-dependent action-potential discharge. However, propofol reduced both the duration and the number of spikes of PDSs recorded from cortical neurons. Interestingly, relatively low concentrations of propofol [half-maximal inhibitory concentration (IC(50)), 3.9 microM) consistently inhibited the "persistent" fraction of Na(+) currents, whereas even high doses (< or =300 microM) had negligible effects on the "fast" component of Na(+) currents. HVA Ca(2+) currents were significantly reduced by propofol, and the pharmacologic analysis of this effect showed that propofol selectively reduced L-type HVA Ca(2+) currents, without affecting N or P/Q-type channels. These results suggest that propofol modulates neuronal excitability by selectively suppressing persistent Na(+) currents and L-type HVA Ca(2+) conductances in cortical neurons. These effects might cooperate with the opening of GABA-A-gated chloride channels, to achieve depression of cortical activity during both anesthesia and status epilepticus.

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca2+-dependent inactivation (CDI) of high-voltage activated (HVA) Ca2+ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca2+-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34+/-9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31+/-3%), a low concentration (3 microM) of parvalbumin (20+/-2%) and calbindin(D28K) (24+/-4%) significantly reduced CDI. Subtype-specific Ca2+ channel blockers indicated that L-type, but not N-type Ca2+ channels are governed by CDI and modulated by Ca2+-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca2+-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22+/-2%; calmodulin binding domain, 17+/-1%) and cytoskeleton stabilizers (taxol, 23+/-5%; phalloidin, 15+/-3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca2+ channels in thalamic physiology.

Melanotrope Cells of Xenopus laevis Express Multiple Types of High‐Voltage‐Activated Ca2+ Channels

Pituitary melanotrope cells are neuroendocrine signal transducing cells that translate physiological stimuli into adaptive hormonal responses. In this translation process, Ca2+ channels play essential roles. We have characterised which types of Ca2+ current are present in melanotropes of the amphibian Xenopus laevis, using whole-cell, voltage-clamp, patch-clamp experiments and specific blockers of the various current types. Running an activation current-voltage relationship protocol from a holding potential (HP) of -80 mV/or -110 mV, shows that Xenopus melanotropes possess only high-voltage activated (HVA) Ca2+ currents. Steady-state inactivation protocols reveal that no inactivation occurs at -80 mV, whereas 30% of the current is inactivated at -30 mV. We determined the contribution of individual channel types to the total HVA Ca2+ current, examining the effect of each channel blocker at an HP of -80 mV and -30 mV. At -80 mV, omega-conotoxin GVIA, omega-agatoxin IVA, nifedipine and SNX-482 inhibit Ca2+ currents by 21.8 +/- 4.1%, 26.1 +/- 3.1%, 24.2 +/- 2.4% and 17.9 +/- 4.7%, respectively. At -30 mV, omega-conotoxin GVIA, nifedipine and omega-agatoxin IVA inhibit Ca2+ currents by 33.8 +/- 3.0, 24.2 +/- 2.6 and 16.0 +/- 2.8%, respectively, demonstrating that these blockers substantially inhibit part of the Ca2+ current, independently from the HP. We have previously demonstrated that omega-conotoxin GVIA can block Ca2+ oscillations and steps. We now show that nifedipine and omega-agatoxin IVA do not affect the intracellular Ca2+ dynamics, whereas SNX-482 reduces the Ca2+ step amplitude. We conclude that Xenopus melanotrope cells express all four major types of HVA Ca2+ channel, as well as the resulting currents, but no low-voltage activated channels. The results provide the basis for future studies on the complex regulation of channel-mediated Ca2+ influxes into this neuroendocrine cell type as a function of its role in the animal's adaptation to external challenges.

The C-terminal Residues in the Alpha-interacting Domain (AID) Helix Anchor CaVβ Subunit Interaction and Modulation of CaV2.3 Channels

The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca(2+) channel alpha1 subunits binds with high affinity to Ca(V)beta auxiliary subunits. The recently solved crystal structures of the AID-Ca(V)beta complex in Ca(V)1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp(6), Leu(7), Gly(9), Tyr(10), Trp(13), and Ile(14) (where the superscript refers to the position of the residue starting with the QQ signature doublet) distributed among three alpha-helical turns in the proximal section of the I-II linker. We show herein that alanine mutations of N-terminal AID residues Gln(1), Gln(2), Ile(3), Glu(4), Glu(6), Leu(7), and Gly(9) in Ca(V)2.3 did not abolish [(35)S]Ca(V)beta 1b or [(35)S]Ca(V)beta 3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of whole cell currents by Ca(V)beta 3. Mutations of the invariant Tyr(10) with either hydrophobic (Ala), aromatic (Phe), or positively charged (Arg, Lys) residues yielded Ca(V)beta 3-responsive whole cell currents, whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca(V)beta 3 binding and modulation. In contrast, modulation and binding by Ca(V)beta 3 was significantly weakened in I14A (neutral and hydrophobic) and I14S (neutral and polar) mutants and eradicated in negatively charged I14D and I14E or positively charged I14R and I14K mutants. Ca(V)beta 3-induced modulation was only preserved with the conserved I14L mutation. Molecular replacement analyses carried out using a three-dimensional homology model of the AID helix from Ca(V)2.3 suggests that a high degree of hydrophobicity and a restrained binding pocket could account for the strict structural specificity of the interaction site found at position Ile(14). Altogether these results indicate that the C-terminal residues Trp(13) (1) and Ile(14) anchor Ca(V)beta subunit functional modulation of HVA Ca(2+) channels.

A cGMP-dependent cascade enhances an l-type-like Ca 2+ current in identified snail neurons

We studied the impact of an NO-cGMP dependent signalling pathway on the high-voltage-activated (HVA) Ca 2+ current in identified neurons of the pulmonate snail, Helix pomatia, using Ba 2+ as charge carrier. The 3′,5′-cyclic guanosine monophosphate (cGMP) analogues, dibutyryl-cGMP and 8-bromo-cGMP, consistently induced a biphasic response, consisting of an increase superseded by a decline of the Ba 2+ current. The NO donor, sodium nitroprusside (SNP), modulated only in a minority of neurons the Ba 2+ current. Blockade of protein kinase activity with 1-[5-isoquinolinesulfonyl]-2 methyl piperazine (H 7), a nonselective protein kinase inhibitor, or Rp-8-pCPT-cGMP, a selective protein kinase G (PKG) inhibitor, decreased, whereas Rp-cAMP, a selective protein kinase A (PKA) inhibitor, increased the Ba 2+ current upon application of cGMP analogues or SNP. Okadaic acid or calyculin, inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), augmented the Ba 2+ current. Under these conditions, cGMP analogues or SNP had an additive-enhancing effect on the Ba 2+ current. When neurons were exposed to the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), cGMP analogues induced a persistent increase of the Ba 2+ current, whereas SNP induced a biphasic response. These data suggest coexistence of cGMP-PKG and cGMP-PDE pathways as well as crosstalk between cGMP and 3′,5′-cyclic adenosine monophosphate (cAMP) pathways, which converge on HVA Ca channels in Helix neurons. In this model, augmentation of the Ba 2+ current through HVA Ca channels is accomplished by PKA and PKG, whereas attenuation is mediated by PDEs, which prevent activation of protein kinases via hydrolysis of cyclic nucleotides.

Angiotensin II inhibition of Ca 2+ currents is independent of ATR1 angiotensin II receptor activation in rat adult vagal afferent neurons

Angiotensin II (ANG II) has the ability to modulate the activity of neurons involved in the cardiovascular regulation. One effective way of doing that is by changing calcium currents. In the present study, we investigated the effects of ANG II on high-voltage-activated (HVA) Ca 2+ currents measured in adult vagal afferent neurons using the whole-cell patch-clamp technique. In addition, we demonstrated the presence of ATR1 and ATR2 receptors mRNA at nodose neurons using conventional reverse transcriptase–polymerase chain reaction (RT–PCR). ANG II (100 nM) decreased the HVA Ca 2+ current (peak current recorded at 0 mV: −60.9±8.7 pA/pF in control conditions versus −31.9±5.7 pA/pF in the presence of ANG II) and shifted the Ca 2+ current activation to a more negative membrane potential (control V 0.5=−12.5±1.5 mV versus −18.4±2.8 mV during perfusion with ANG II). Losartan (500 nM) was not able to prevent the ANG II effect on the HVA Ca 2+ current making unlikely the involvement of the ATR1 receptor. When ANG II was perfused in the continuous presence of saralasin, a non-selective ANG II receptor antagonist, we observed a faster but transient inhibition of HVA Ca 2+ current. The inhibition was not sustained as observed when we applied ANG II alone and the HVA Ca 2+ current recovered with time reaching levels close to the control. Unexpectedly, treatment with the ATR2 blocker PD 123,319 (500 nM) caused a significant inhibition on the HVA Ca 2+ current making rather difficult any further conclusions. The above results allow us to conclude that ANG II induced inhibition on the HVA Ca 2+ current is probably not via ATR1 receptor activation.

High voltage-activated Ca 2+ channel currents in isolectin B 4-positive and -negative small dorsal root ganglion neurons of rats

Voltage-gated Ca 2+ channels in the primary sensory neurons are important for neurotransmitter release and regulation of nociceptive transmission. Although multiple classes of Ca 2+ channels are expressed in the dorsal root ganglion (DRG) neurons, little is known about the difference in the specific channel subtypes among the different types of DRG neurons. In this study, we determined the possible difference in high voltage-activated Ca 2+ channel currents between isolectin B 4 (IB 4)-positive and IB 4-negative small-sized (15–30 μm) DRG neurons. Rat DRG neurons were acutely isolated and labeled with IB 4 conjugated to a fluorescent dye. Whole-cell patch clamp recordings of barium currents flowing through calcium channels were performed on neurons with and without IB 4. The peak current density of voltage-gated Ca 2+ currents was not significantly different between IB 4-positive and IB 4-negative neurons. Also, both nimodipine and ω-agatoxin IVA produced similar inhibitory effects on Ca 2+ currents in these two types of neurons. However, block of N-type Ca 2+ channels with ω-conotoxin GVIA produced a significantly greater reduction of Ca 2+ currents in IB 4-positive than IB 4-negative neurons. Furthermore, the IB 4-positive neurons had a significantly smaller residual Ca 2+ currents than IB 4-negative neurons. These data suggest that a higher density of N-type Ca 2+ channels is present in IB 4-positive than IB 4-negative small-sized DRG neurons. This differential expression of the subtypes of high voltage-activated Ca 2+ channels may contribute to the different function of these two classes of nociceptive neurons.

Differential sensitivity of N- and P/Q-type Ca2+ channel currents to a mu opioid in isolectin B4-positive and -negative dorsal root ganglion neurons.

Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to isolectin B4(IB4). In this study, we determined the difference in the modulation of voltage-gated Ca2+ currents by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a specific mu opioid agonist) between IB4-positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1-10 microM DAMGO and 1 to 10 microM morphine had a greater effect on high voltage-activated Ca2+ currents in IB4-negative than IB4-positive cells. However, DAMGO had no significant effect on T-type Ca2+ currents in both groups. The N-type Ca2+ current was the major subtype of Ca2+ currents inhibited by DAMGO in both IB4-positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca2+ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca2+ currents in IB4-negative than IB4-positive neurons. Furthermore, double labeling revealed that there was a significantly higher mu opioid receptor immunoreactivity in IB4-negative than IB4-positive cells. Thus, these data suggest that N-and P/Q-type Ca2+ currents are more sensitive to inhibition by the mu opioids in IB4-negative than IB4-positive DRG neurons. The differential sensitivity of voltage-gated Ca2+ channels to the mu opioids in subsets of DRG neurons may constitute an important analgesic mechanism of mu opioids.

Modulation of ion channels and synaptic transmission by a human sensory neuron-specific G-protein-coupled receptor, SNSR4/mrgX1, heterologously expressed in cultured rat neurons.

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8-22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca2+ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca2+ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K+ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8-22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to G(q/11)-proteins as well as PTX-sensitive G(i/o)-proteins, and modulates neuronal Ca2+ channels, K+ channels, and synaptic transmission.

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha.

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 diabetes.

Multiple Structural Elements Contribute to the Slow Kinetics of the Cav3.3 T-type Channel

Molecular cloning and expression studies established the existence of three T-type Ca(2+) channel (Ca(v)3) alpha(1) subunits: Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I)). Although all three channels are low voltage-activated, they display considerable differences in their kinetics, with Ca(v)3.1 and Ca(v)3.2 channels activating and inactivating much faster than Ca(v)3.3 channels. The goal of the present study was to determine the structural elements that confer the distinctively slow kinetics of Ca(v)3.3 channels. To address this question, a series of chimeric channels between Ca(v)3.1 and Ca(v)3.3 channels were constructed and expressed in Xenopus oocytes. Kinetic analysis showed that the slow activation and inactivation kinetics of the Ca(v)3.3 channel were not completely abolished by substitution with any one portion of the Ca(v)3.1 channel. Likewise, the Ca(v)3.1 channel failed to acquire the slow kinetics by simply adopting one portion of the Ca(v)3.3 channel. These findings suggest that multiple structural elements contribute to the slow kinetics of Ca(v)3.3 channels.

Calcium influx through presynaptic 5-HT 3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies

Serotonin 5-hydroxytryptamine type 3 receptors (5HT 3R) are Ca 2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [ 3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT 3Rs, as it was mimicked by the selective 5HT 3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT 3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT 3R-mediated increase in GABA release was blocked by 100 μM cadmium or by omitting Ca 2+ in external solutions, indicating the Ca 2+-dependence of the effect. The high voltage-activated Ca 2+ channel blockers ω-conotoxin GVIA and ω-conotoxin MVIIC and 10 μM cadmium had no significant effect on the 5-HT 3R-mediated enhancement of GABA release, indicating that Ca 2+ influx through the 5-HT 3R facilitates GABA release. Taken together, these data provide direct evidence that Ca 2+ entry via presynaptic 5HT 3Rs facilitates the release of GABA from hippocampal interneurons.

Targeting mechanisms of high voltage-activated Ca2+ channels.

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

Functional expression of L- and T-type Ca 2+ channels in murine HL-1 cells

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca 2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca 2+ channels including the L-types, α1C and α1D, as well as T-types, α1H and α1G, but are lacking N-type, α1B and the T-type, α1I. Western blot analysis demonstrated significant α1C protein subunit expression, with less α1D subunit apparent, while α1A, α1B and α1E subunit expression was undetectable. Immunocytochemical staining showed that the α1C protein subunit is expressed predominantly on the cell surface, whereas the α1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low ( I Ca,T) and high ( I Ca,L) voltage-activated Ca 2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca 2+ concentrations, [Ca 2+] o, resulted in Ca 2+ influx measured by fluorometric imaging with an EC 50 of 0.8 mM [Ca 2+] o. At a fixed [Ca 2+] o of 0.125 mM, Ca 2+ influx was also triggered by increasing the extracellular K + concentration, [K +] o, with an EC 50 of 3.7 mM [K +] o. As increasing [K +] o depolarizes the cell, this latter result is consistent with Ca 2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca 2+ channel blockers inhibited Ca 2+ influx with IC 50s of 1, 2, 0.4 and 0.2 μM, respectively. Antagonists of N-type (ω-conotoxins GVIA) and P/Q-type (MVIIC or ω-agatoxin IVA) did not inhibit Ca 2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca 2+ channels.

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure ( pO 2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca 2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO 2 to 15–30 mm Hg induced a potentiation of HVA Ca 2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca 2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons.

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ ( IBa) with IC50 of approximately 920 nM, approximately 58 micro M, and approximately 65 micro M, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.