HVA Calcium Research Articles

Inhibition of high-voltage-activated calcium currents by acute hypoxia in cultured retinal ganglion cells.

Hypoxia is a common factor of numerous ocular diseases that lead to dysfunctions and loss of retinal ganglion cells (RGCs) with subsequent vision loss. High-voltage-activated calcium channels are the main source of calcium entry into neurons. Their activity plays a central role in different signaling processes in health and diseases, such as enzyme activation, gene transcription, synaptic transmission, or the onset of cell death. This study aims to establish and evaluate the initial effect of the early stage of acute hypoxia on somatic HVA calcium currents in cultured RGCs. HVA calcium currents were recorded in RGCs using the whole-cell patch-clamp technique in the voltage-clamp mode. The fast local superfusion was used for a brief (up to 270 s) application of the hypoxic solution (pO2 < 5 mmHg). The switch from normoxic to hypoxic solutions and vice versa was less than 1 s. The HVA calcium channel activity was inhibited by acute hypoxia in 79% of RGCs (30 of 38 RGCs) in a strong voltage-dependent manner. The level of inhibition was independent of the duration of hypoxia or repeated applications. The hypoxia-induced inhibition of calcium currents had a strong correlation with the duration of hypoxia and showed the transition from reversible to irreversible at 75 s of hypoxia and longer. The results obtained are the first demonstration of the phenomena of HVA calcium current inhibition by acute hypoxia in RGCs and provide a conceptual framework for further research.

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells’ current kinetics and current–voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV) Channels.

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1–1.4; CaV2.1–2.3) and Low-voltage activated (LVA, CaV3.1–3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, “conopeptides”, which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

Impact of gabapentin on neuronal high voltage-activated Ca2+ channel properties of injured-side axotomized and adjacent uninjured dorsal root ganglions in a rat model of spinal nerve ligation.

The density and properties of ion channels in the injured axon and dorsal root ganglion (DRG) neuronal soma membrane change following nerve injury, which may result in the development of neuropathic pain. Gabapentin (GBP) is a drug for the first-line treatment of neuropathic pain. One of its therapeutic targets is the voltage-activated calcium channel (VACC). In the present study, the whole-cell patch clamp technique was used to examine the changes of high voltage-activated Ca2+ (HVA-Ca2+) channels in DRG neurons from sham and neuropathic rats in the absence and presence of GBP. The results demonstrated a reduction in peak current density and the ‘window current’ between activation and inactivation in adjacent and axotomized neurons from rats that had undergone L5 spinal nerve ligation, thus attenuating the total inward Ca2+ current. Following the use of the specific channel blockers nifedipine, ω-conotoxin MVIIC and ω-conotoxin MVIIA, increased HVA-Ca2+ channels as well as an increased proportion of N-type Ca2+ currents were observed in axotomized neurons. GBP inhibited HVA calcium channel currents in a dose-dependent manner. The activation and steady-state inactivation curves for HVA channels were shifted in a hyperpolarizing direction by 100 µmol/l GBP. Following the application of GBP, a reduction in the ‘window current’ was observed in control and axotomized neurons, whereas the ‘window current’ was unchanged in adjacent neurons. This indicates that the inhibitory effects of GBP may be dependent on particular neuropathological or inflammatory conditions. The proportion of N-type Ca2+ currents and sensitivity to GBP were increased in axotomized neurons, which indicated the involvement of N-type Ca2+ currents in the inhibitory effect of GBP.

The Kelch‐Like 1 Actin Binding Protein Modulates Neuronal Voltage Gated Calcium Channels

Kelch‐like 1 (KLHL1) is a neuronal actin‐binding protein first identified in humans as part of the genetic locus of the neurological movement disorder Spinocerebellar Ataxia type 8. KLHL1 is expressed in the cytosol, axons and dendrites. KLHL1 KO causes loss of post‐synaptic structures, loss of motor coordination and gait abnormalities. KLHL1 is an ion channel modulator and upregulates HVA and LVA calcium currents in vitro by increased recycling endosome activity and by alteration of the kinetics of τdeactivation. We analyzed the endogenous HVA and LVA calcium currents in neurons from WT and KLHL1 KO mice, and compared these data with the acute down‐regulation of KLHL1 using a knock‐down shRNA (KD) strategy. KLHL1 KD caused decreased HVA and LVA calcium current expression. In contrast, the KO had no differences in total LVA current density, yet changes in current kinetics suggest there is a down‐regulation of α1H and compensatory expression of α1I. HVA current density was also decreased in KO (40%) at 4–6 DIV but not at 7–10 DIV, suggesting this deficit was also compensated for after 7 DIV. Our data confirm that KLHL1 modulates LVA and HVA channel function in vivo; the compensatory modulation of currents in the KO may contribute to the mild motor phenotype seen in these mice.This material is based upon work supported by the National Science Foundation under Grant No. 1022075

Effects of Cannabinoids on Ion Channels of Pancreatic Beta Cells

The function of the beta cell of the pancreatic islet is to sense minute changes in blood glucose levels and secrete insulin to maintain euglycemia. Though this secretion is governed chiefly by glucose, other chemical factors such as insulin itself and the incretin hormones also modulate this function. Peliminary evidence in our lab indicates that endocannabinoids are important negative regulators of insulin secretion. Beta cells are electrically excitable and undergo depolarization upon glucose stimulated insulin secretion resulting from closure of the K(ATP) channels. As a consequence, the calcium channels open, intracellular levels of calcium rise and exocytosis of the insulin secretory granules occurs. In addition to the K(ATP) channels and the calcium channels, beta cells also possess an array of ion channels that generate the complex electrical waveforms and participate to varying degrees in the regulation of the membrane potential.Using rodent beta cells we investigated the effects of cannabinoids on some of these ion channels. Thus, 10 μM 2-arachidonylglycerol (2-AG) decreased the amplitude of the delayed rectifier current by about 40%. Simple washing did not reverse this blockade, and the addition of the cannabinoid receptor 1 (CB1) antagonist AM 251 (1 μ) also had no effect. However, the blockade was completely reversed by washing with lipid free bovine serum albumin. Moreover, the CB1 agonist, WIN-55,212-2(1 μM ), an indole derivative structurally unrelated to the lipid 2-AG, had no antagonistic effect on the delayed rectifier. Taken together, these results suggest that the block was independent of the CB1 receptor and was mediated instead via the plasma membrane. Cannabinoid blockade of both sodium and HVA calcium currents were similar to the effects on the delayed rectifier.

Role of voltage-gated calcium channels in epilepsy

It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high- and low-voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 alpha1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to co-assemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits, there is a compensatory increase in thalamic LVA currents which likely leads to the seizure phenotype. In fact, gain-of-function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. In this paper, we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.

Real-time imaging of calcium dynamics in rhythmically active neurons of thespinal network for locomotion

Event Abstract Back to Event Real-time imaging of calcium dynamics in rhythmically active neurons of thespinal network for locomotion Di Wang1*, Sten Grillner1 and Peter Wallén1 1 Karolinska Institutet, Sweden The neuronal network underlying locomotion in vertebrates has been investigated in considerable detail in the lamprey spinal cord in vitro model. The isolated spinal cord preparation can be made to produce well-coordinated rhythmic activity, with left-right alternation and intersegmental phase-coupling, corresponding to locomotion (swimming). The rhythm-generating capacity of the network is the result of an interplay between network connectivity properties and the dynamic membrane properties of the individual network neurons. These different kinds of membrane properties typically rely on localized influx of calcium, leading to, for instance, the activation of calcium-dependent potassium channels (KCa-channels). Among the key calcium-dependent membrane properties are the NMDA-receptor-dependent membrane potential pacemaker oscillations, the slow afterhyperpolarization (sAHP) following the action potential, and the postinhibitory rebound depolarization following a period of hyperpolarization. Although much is known about the detailed operation of the network, specific information on the calcium dynamics – both in the temporal and the spatial domain – is still to a large degree lacking. We are therefore investigating different membrane properties of lamprey spinal neurons by utilizing confocal calcium imaging in combination with intracellular membrane potential recordings. During rhythmic network operation (fictive locomotion), calcium fluctuations can be detected in dendrites of individual neurons, even during subthreshold membrane potential oscillations (cf. Bacskai et al., Neuron 14:19-28, 1995). Furthermore, during NMDA-dependent pacemaker oscillations in the presence of TTX, dendritic calcium fluctuations are also seen, timed to the membrane potential oscillations (Fig. 1). During burst firing, the sAHP´s following each spike will add and produce a pronounced summed sAHP, which contributes to the termination of the burst. Calcium imaging during spike trains has revealed a distinct influx of calcium in conjunction with the sAHP. This calcium enters via N (CaV 2.2)- and P/Q (CaV 2.1) types of HVA calcium channels, while L-type channels (CaV 1) are not involved. Correspondingly, blockade of N- and P/Q-type channels reduced the calcium influx in the soma and proximal dendrites, while blockade of L-type channels did not. This indicates that the calcium channels responsible for activating the KCa channels involved in the sAHP are primarily located in the soma and proximal dendrites of the cell. In contrast, L-type calcium channels appear to be mainly localized to distal dendrites in lamprey spinal neurons. Confocal fluorescence imaging of calcium influx, and other dynamic events, with high spatial and temporal resolution, in combination with electrophysiological recordings from the same neuron in the functional network, holds promise to reveal important insights into the cellular and subcellular mechanisms governing network operation. tn_image2 conference image2 conference Conference: Neuroinformatics 2008, Stockholm, Sweden, 7 Sep - 9 Sep, 2008. Presentation Type: Poster and Short Oral Presentation Topic: Neuroimaging Citation: Wang D, Grillner S and Wallén P (2008). Real-time imaging of calcium dynamics in rhythmically active neurons of thespinal network for locomotion. Front. Neuroinform. Conference Abstract: Neuroinformatics 2008. doi: 10.3389/conf.neuro.11.2008.01.060 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Jul 2008; Published Online: 25 Jul 2008. * Correspondence: Di Wang, Karolinska Institutet, Stockholm, Sweden, di.wang@ki.se Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Di Wang Sten Grillner Peter Wallén Google Di Wang Sten Grillner Peter Wallén Google Scholar Di Wang Sten Grillner Peter Wallén PubMed Di Wang Sten Grillner Peter Wallén Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Inhibition of insect calcium channels by huwentoxin-V, a neurotoxin from Chinese tarantula Ornithoctonus huwena venom

The effects of huwentoxin-V, an insect neurotoxic peptide from Chinese tarantula Ornithoctonus huwena venom, were studied on neuronal voltage-gated ion channels. Whole-cell patch-clamp configuration indicated that huwentoxin-V specifically inhibited high-voltage-activated calcium channels in adult cockroach dorsal unpaired median neurons (IC 50 ≈ 219 nM) while having no evident effect on voltage-gated potassium and sodium channels. Omega-conotoxin GVIA is a well-known neuronal N-type calcium channel blocker from the venom of the sea snail Conus geographus and it also can partially block calcium currents in cockroach dorsal unpaired median neurons. In our study, huwentoxin-V inhibited omega-conotoxin GVIA-sensitive, diltiazem-sensitive and partial omega-conotoxin GVIA and diltiazem-resistant calcium currents elicited from insect neurons. Based on the sensitivity of calcium currents to these toxins, insect neuronal HVA calcium channels might be classified into four types: Type I, omega-conotoxin GVIA-sensitive and huwentoxin-V-sensitive; type II, diltiazem-sensitive and huwentoxin-V-sensitive; type III, huwentoxin-V-sensitive but omega-conotoxin GVIA and diltiazem-resistant; type IV, omega-conotoxin GVIA and diltiazem-resistant and huwentoxin-V-resistant. Its specificity, incomplete inhibition and insect-toxic effects make it an interesting tool for investigating insect voltage-gated calcium channels and development of new insecticidal compounds.

A short history of voltage‐gated calcium channels

British Journal of Pharmacology (2006) 147, S56–S62. doi:10.1038/sj.bjp.0706442

A single CaVβ can reconstitute both trafficking and macroscopic conductance of voltage‐dependent calcium channels

Voltage-dependent calcium-channel beta subunits (Ca(V)beta) strongly modulate pore-forming alpha(1) subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P(o)). Despite their central role, it is unclear whether binding of a single Ca(V)beta, or multiple Ca(V)betas, to an alpha(1) subunit governs the two distinct functions. Conventional experiments utilizing coexpression of alpha(1) and Ca(V)beta subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1: 1 stoichiometry by covalently linking Ca(V)beta(2b) to the carboxyl terminus of alpha(1C) (Ca(V)1.2), creating alpha(1C).beta(2b). Recombinant L-type channels reconstituted in HEK 293 cells with alpha(1C).beta(2b) supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed alpha(1C).beta(2b) fully restored channel trafficking to the plasma membrane. Co-transfecting Ca(V)beta(2a) with alpha(1C).beta(2b) had little further impact on function. To rule out the possibility that fused Ca(V)beta(2b) was interacting in trans with neighbouring alpha(1) molecules, alpha(1C).beta(2b) was cotransfected with alpha(1B) (Ca(V)2.2), and pharmacological block with nimodipine showed an absence of alpha(1B) trafficking. These results establish that association of a single Ca(V)beta with a pore-forming alpha(1) subunit captures the functional essence of HVA calcium channels, and introduce alpha(1)-Ca(V)beta fusion proteins as a powerful new tool to probe structure-function mechanisms.

Postnatal Development Differentially Affects Voltage-Activated Calcium Currents in Respiratory Rhythmic Versus Nonrhythmic Neurons of the Pre-Bötzinger Complex

The mammalian respiratory network reorganizes during early postnatal life. We characterized the postnatal developmental changes of calcium currents in neurons of the pre-Bötzinger complex (pBC), the presumed site for respiratory rhythm generation. The pBC contains not only respiratory rhythmic (R) but also nonrhythmic neurons (nR). Both types of neurons express low- and high-voltage-activated (LVA and HVA) calcium currents. This raises the interesting issue: do calcium currents of the two co-localized neuron types have similar developmental profiles? To address this issue, we used the whole cell patch-clamp technique to compare in transverse slices of mice LVA and HVA calcium current amplitudes of the two neuron populations (R and nR) during the first and second postnatal week (P0-P16). The amplitude of HVA currents did not significantly change in R pBC-neurons (P0-P16), but it significantly increased in nR pBC-neurons during P8-P16. The dehydropyridine (DHP)-sensitive current amplitudes did not significantly change during the early postnatal development, suggesting that the observed amplitude changes in nR pBC-neurons are caused by (DHP) insensitive calcium currents. The ratio between HVA calcium current amplitudes dramatically changed during early postnatal development: At P0-P3, current amplitudes were significantly larger in R pBC-neurons, whereas at P8-P16, current amplitudes were significantly larger in nR pBC-neurons. Our results suggest that calcium currents in pBC neurons are differentially altered during postnatal development and that R pBC-neurons have fully expressed calcium currents early during postnatal development. This may be critical for stable respiratory rhythm generation in the underlying rhythm generating network.

Ca(v)3.2 calcium channels control an autocrine mechanism that promotes neuroblastoma cell differentiation.

Calcium influx via low-voltage activated alpha(1H) (Ca(v)3.2) T-currents participates in the morphological and electrical differentiation of neuroblastoma NG108-15 cells. We investigated whether an autocrine mechanism could contribute to this differentiation process. The presence of factors secreted by NG108-15 cells was identified through the use of conditioned media (CM) obtained from differentiated cells. These CM significantly increased neuritogenesis with no change in the HVA calcium channel expression. CM-induced neuritogenesis persists during alpha(1H) current block, whereas CM obtained from cells transfected with an alpha(1H) antisense did not induce neuritogenesis. These data indicate that morphological differentiation of NG108-15 cells depends on an autocrine mechanism, which is controlled by alpha(1H) currents. Such a mechanism is likely to play a role in the various differentiation processes that imply alpha(1H) T-type Ca(2+) channels.

Distribution and relative expression levels of calcium channel β subunits within the chambers of the rat heart

Calcium channel β subunits expressed in rat atria and atrial myocytes are identified and their expression quantified and compared to β subunit expression in the ventricle. mRNAs encoding all four know β subunits are expressed in atrial myocytes including the following splice variants: β 1a, β 2b, β 2c, β 2e and β 4d. The specific β 2 splice variants expressed in the atria (β 2b, β 2c, β 2e) differ from those previously reported from rat ventricle. β 2 isoform is the most abundant β mRNA expressed in the heart and the amount of both β 2 subunit mRNA and β 2 subunit protein is significantly greater in the ventricles than in the atria. The expression of individual β 2 splice variants varies with age and within different chambers of the heart. The β 2b splice variant appears in both atria and ventricle in both young (4.5 week) and old (16 week) animals, the β 2c isoform is more highly expressed in young as compared to old animals and the β 2e splice variant is robustly expressed only in 4.5 week ventricle. β 4 mRNA expression is higher in atrial tissue than in ventricles and its expression decreases in older animals. In contrast, the abundance of the β 3 mRNA does not significantly change as a function of postnatal age. Antisense oligonucleotides targeting sequence common to all the β isoforms as well as that specific for β 2 significantly reduced HVA calcium current density in isolated atrial cells confirming that the β 2 subunits contribute to the regulation of HVA calcium current expression in the rat atria. The complexity of β isoform expression within the heart may provide a mechanism for functional fine-tuning of the cardiac HVA current.

Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels.

Voltage-dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high-voltage-activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore-lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I-II linker region acts as a 'hinged lid' that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel Beta subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III-IV linker regions of the channel modulate inactivation rates through interactions with the I-II linker per se, or indirectly via the ancillary Beta subunits. Taken together, the fast voltage-dependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.

Modulation by extracellular pH of low- and high-voltage-activated calcium currents of rat thalamic relay neurons.

The effects of changes in the extracellular pH (pH(o)) on low-voltage- (LVA) and high-voltage- (HVA) activated calcium currents of acutely isolated relay neurons of the ventrobasal thalamic complex (VB) were examined using the whole cell patch-clamp technique. Modest extracellular alkalinization (pH 7.3 to 7.7) reversibly enlarged LVA calcium currents by 18.6 +/- 3.2% (mean +/- SE, n = 6), whereas extracellular acidification (pH 7.3 to 6.9) decreased the current by 24.8 +/- 3.1% (n = 9). Normalized current amplitudes (I/I(7.3)) fitted as a function of pH(o) revealed an apparent pK(a) of 6.9. Both, half-maximal activation voltage and steady-state inactivation were significantly shifted to more negative voltages by 2-4 mV on extracellular alkalinization and to more positive voltages by 2-3 mV on extracellular acidification, respectively. Recovery from inactivation of LVA calcium currents was not significantly affected by changes in pH(o). In contrast, HVA calcium currents were less sensitive to changes in pH(o). Although extracellular alkalinization increased maximal HVA current by 6.0 +/- 2.0% (n = 7) and extracellular acidification decreased it by 11.9 +/- 0.02% (n = 11), both activation and steady-state inactivation were only marginally affected by the moderate changes in pH(o) used in the present study. The results show that calcium currents of thalamic relay neurons exhibit different pH(o) sensitivity. Therefore activity-related extracellular pH transients might selectively modulate certain aspects of the electrogenic behavior of thalamic relay neurons.

Ca(2+) channels involved in the generation of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurons.

The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM) and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 microM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.

Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons.

The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P¿Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.

The serotonin inhibition of high-voltage-activated calcium currents is relieved by action potential-like depolarizations in dissociated cholinergic nucleus basalis neurons of the guinea-pig.

The aim of the present study was to investigate whether the voltage-dependent inhibition of calcium currents by serotonin 5-HT1A agonists can be alleviated (facilitated) by action potential-like depolarizations. In dissociated cholinergic basal forebrain neurons using whole-cell recordings, it is shown that a selective serotonin 5-HT1A agonist (8-OH-DPAT) predominantly blocks N-type HVA calcium current, although a minor reduction of P-type current was also observed. The inhibition may principally occur through Gi-Go subtypes of G-proteins because it was prevented by N-ethylmaleimide, a substance known to block specifically pertussis-sensitive G-proteins. The inhibitory effect of 8-OH-DPAT on calcium currents is voltage-dependent because it was alleviated by long-lasting depolarizing prepulses. Interestingly, the inhibition could also be reversed by prepulses made-up of action potential-like depolarizations that were given at a frequency of 200 Hz. This observation may have important implications during periods of high-frequency rhythmic bursts, a firing pattern that is prevalent in cholinergic basal forebrain neurons.

Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex.

A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 +/- 6.2 (SD) pF] than in neurons from layers II and III (8.6 +/- 2.8 and 7.9 +/- 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to -40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11-16 pA/pF at -30 mV, 2) inactivated rapidly with a time constant of approximately 22 ms at -30 mV, and 3) was completely steady-state inactivated at -60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.