Low Voltage-activated Ca2 Research Articles

Potent inhibition of a recombinant low voltage-activated Ca 2+ channel by SB-209712

T-type Ca 2+ currents were recorded in 2 mM Ca 2+ from HEK293 cells stably expressing the low voltage-activated Ca 2+ channel sub-unit α 1I. These currents were inhibited by the known Ca 2+ channel antagonist mibefradil with an IC 50 close to 1 μM. SB-209712 (1,6,bis{1-[4-(3-phenylpropyl)piperidinyl]}hexane), a compound originally developed as a high voltage-activated Ca 2+ channel blocker, proved to be a more potent T-type channel antagonist, exhibiting an IC 50 in the region of 500 nM. The antagonism produced by SB-209712 was reversed following drug removal and the observed antagonism exhibited little or no voltage-dependence with respect to either holding or test potential. These data indicate that SB-209712 is amongst the most potent known non-peptide T-type channel antagonists and thus may have some use in understanding the role of these channels in cellular function.

γ-Tocopherol Partially Protects Insulin-Secreting Cells against Functional Inhibition by Nitric Oxide

Preceding the onset of type 1 diabetes mellitus, pancreatic islets are infiltrated by macrophages secreting interleukin-1β (IL-1β) which induces β-cell apoptosis and exerts inhibitory actions on islet β-cell insulin secretion. IL-1β seems to act chiefly through induction of nitric oxide (NO) synthesis. Hence, IL-1β and NO have been implicated as key effector molecules in type 1 diabetes mellitus. In this paper, the influence of endogenously produced and exogenously delivered NO on the regulation of cell proliferation, cell viability and discrete parts of the stimulus-secretion coupling in insulin-secreting RINm5F cells was investigated. Because vitamin E may delay diabetes onset in animal models, we also investigated whether tocopherols may protect β-cells from the suppressive actions of IL-1 and NO in vitro. To this end, the impact of NO on insulin secretory responses to activation of phospholipase C (by carbamylcholine), protein kinase C (by phorbol ester), adenylyl cyclase (by forskolin), and Ca2+ influx through voltage-activated Ca2+ channels (by K+-induced depolarization) was monitored in culture after treatment with IL-1β or by co-incubation with the NO donor spermine-NONOate. It was found that cell proliferation, viability, insulin production and the stimulation of insulin release evoked by carbamylcholine and phorbol ester were impeded by IL-1β or spermine-NONOate, whereas the hormone output by the other secretagogues was not altered by NO. Pretreatment with γ-tocopherol (but not α-tocopherol) afforded a partial protection against the inhibitory effects of NO, whereas specifically inhibiting inducible NO synthase with N-nitro-L-arginine completely reversed the IL-1β effects. In contrast, inhibiting guanylyl cyclase with ODQ (1H-[1,2,4]oxadiazolo[4,3-α]-quinoxaline-1-one) or blocking low voltage-activated Ca2+ channels with NiCl2 failed to influence the actions of NO. In conclusion, our data show that NO inhibits growth and insulin secretion in RINm5F cells, and that γ-tocopherol may partially prevent this. The results suggest that phospholipase C or protein kinase C may be targeted by NO. In contrast, cGMP or low voltage-activated Ca2+ channels appear not to mediate the toxicity of NO in these cells. These adverse effects of NO on the β-cell, and the protection by γ-tocopherol, may be of importance for the development of the impaired insulin secretion characterizing type 1 diabetes mellitus, and offer possibilities for intervention in this process.

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels.

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.

Pharmacological properties of Ca(V)3.2, a low voltage-activated Ca2+ channel cloned from human heart.

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.

Na+ currents through Ca2+ channels in human retinal glial (M�ller) cells

To detect the presence of voltage-gated Ca(2+) channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA(B) receptor agonists. Whole cell voltage-clamp recordings were made to study Ca( 2+), Ba(2+), and Na(+) currents through voltage-gated Ca(2+) channels. The vast majority of the investigated cells displayed no resolvable currents through Ca(2+) channels when Ca(2+) ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents ( approximately 0.6 pA/pF) were detected when Ba(2+) ions (20 mM) were used as charge carrier. However, when Na(+) ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes ( approximately 7.5 pA/pF) through voltage-gated Ca(2+) channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca(2+) channels and long-lasting, high voltage-activated channels. The Na(+) fluxes through low voltage-activated Ca( 2+) channels were inhibited in a voltage-independent manner in the presence of GABA(B) receptor agonists. Human Müller glial cells express different kinds of voltage-gated Ca(2+) channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na(+) fluxes in divalent cation-free solutions may be a technique to detect the presence of "hidden" voltage-gated Ca(2+) channels in Müller glial cells.

Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells.

1. The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. 2. In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25-50 microM), a blocker of low voltage-activated T-type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. 3. Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about -60 mV. Ni2+ suppressed low voltage-activated Ca2+ release. Moreover, low voltage-activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+-Ca2+ exchange (INa-Ca). Low voltage-activated Ca2+ release was found in both sino-atrial node and latent atrial pacemaker cells but not in working atrial myocytes. 4. These findings suggest that low voltage-activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa-Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.

Differential expression of high- and two types of low-voltage-activated calcium currents in rod and cone bipolar cells of the rat retina.

Whole cell voltage-clamp recordings were performed to investigate voltage-activated Ca(2+) currents in acutely isolated retinal bipolar cells of rats. Two groups of morphologically different bipolar cells were observed. Bipolar cells of the first group, which represent the majority of isolated bipolar cells, were immunoreactive to protein kinase C (PKC) and, therefore likely to be rod bipolar cells. Bipolar cells of the second group, which represent only a small population of isolated bipolar cells, did not show PKC immunoreactivity and were likely to be cone bipolar cells. The validity of morphological identification of bipolar cells was further confirmed by the presence of GABA(C) responses in these cells. Bipolar cells of both groups displayed low-voltage-activated (LVA) Ca(2+) currents with similar voltage dependence of activation and steady-state inactivation. However, the activation, inactivation, and deactivation kinetics of the LVA Ca(2+) currents between rod and cone bipolar cells differed. Particularly, the LVA Ca(2+) currents of rod bipolar cells displayed both transient and sustained components. In contrast, the LVA Ca(2+) currents of cone bipolar cells were mainly transient. In addition, the LVA Ca(2+) channels of rod bipolar cells were more permeable to Ba(2+) than to Ca(2+), whereas those of cone bipolar cells were equally or less permeable to Ba(2+) than to Ca(2+). The LVA Ca(2+) currents of both rod and cone bipolar cells were antagonized by high concentrations of nimodipine with IC(50) of 17 and 23 microM, respectively, but largely resistant to Cd(2+) and Ni(2+). Bipolar cells of both groups also displayed high-voltage-activated (HVA) Ca(2+) currents. The HVA Ca(2+) currents were, at least in part, to be L-type that were potentiated by BayK-8644 (1 microM) and largely antagonized by low concentrations of nimodipine (5 microM). The L-type Ca(2+) channels were almost exclusively located at the axon terminals of rod bipolar cells but expressed at least in the cell soma of cone bipolar cells. Results of this study indicate that rod and cone bipolar cells of the mammalian retina differentially express at least two types of LVA Ca(2+) channels. Rod and cone bipolar cells also show different spatial distribution of L-type Ca(2+) channels.

Na+ currents through Ca2+ channels in human retinal glial (Müller) cells

Purpose. To detect the presence of voltage-gated Ca 2+ channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA B receptor agonists. Methods. Whole cell voltage-clamp recordings were made to study Ca 2+, Ba 2+, and Na + currents through voltage-gated Ca 2+ channels. Results. The vast majority of the investigated cells displayed no resolvable currents through Ca 2+ channels when Ca 2+ ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents (~0.6 pA/pF) were detected when Ba 2+ ions (20 mM) were used as charge carrier. However, when Na + ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes (~7.5 pA/pF) through voltage-gated Ca 2+ channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca 2+ channels and long-lasting, high voltage-activated channels. The Na + fluxes through low voltage-activated Ca 2+ channels were inhibited in a voltage-independent manner in the presence of GABA B receptor agonists. Conclusions. Human Müller glial cells express different kinds of voltage-gated Ca 2+ channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na + fluxes in divalent cation-free solutions may be a technique to detect the presence of “hidden” voltage-gated Ca 2+ channels in Müller glial cells.

Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation.

Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca2+ entry into sperm through low voltage-activated Ca2+ channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca2+ channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca2+ levels were used to demonstrate that (i) contact with zonae pellucidae activates a transient Ca2+ response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca2+ channels; (ii) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (iii) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (iv) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca2+ channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.

Cloning and Expression of a Novel Member of the Low Voltage-Activated T-Type Calcium Channel Family

Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

A low voltage-activated Ca2+ current mediates cytokine-induced pancreatic beta-cell death.

Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic beta-cells. Chronic treatment with cytokines induced a low voltage-activated (LVA) Ca2+ current in mouse beta-cells. The concomitant increase in the basal cytoplasmic free Ca2+ concentration ([Ca2+]i) was associated with DNA fragmentation and cell death. Antagonists of LVA Ca2+ channels prevented this elevation of basal [Ca2+]i and DNA fragmentation and reduced the percentage of cell death. Exposure to cytokines did not affect the profile of Ca2+ currents or basal [Ca2+]i in glucagon-secreting alpha-cells. An increased Ca2+ signal through LVA Ca2+ channels may thus be a key feature in cytokine-induced beta-cell destruction.

A Low Voltage-Activated Ca2+ Current Mediates Cytokine-Induced Pancreatic -Cell Death

A Low Voltage-Activated Ca2+ Current Mediates Cytokine-Induced Pancreatic -Cell Death

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (INa(V)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of INa(V). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. ω-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 191–206, 1999

Ion conductances related to development of repetitive firing in mouse retinal ganglion neuronsin situ

In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.

Divalent cation selectivity of the subtypes of low voltage-activated Ca2+ channels in thalamic neurons.

LVA Ca2+ current in isolated associative neurons from the laterodorsal thalamic nucleus of 14- to 17-day-old rats was dissected into two, 'fast' and 'slow', components based on the difference in the kinetics of inactivation. The selectivity of the channel responsible for the fast LVA current for Ca2+, Sr2+ and Ba2+ (I(Ca):I(Sr):I(Ba) = 1.0:1.23:0.94) as well as the shifts of the I-V produced by these ions were found to be almost identical to those observed for LVA channels in other preparations. The channel responsible for the slow LVA current showed selectivity more characteristic of HVA Ca2+ channels (I(Ca):I(Sr):I(Ba) = 1.0:2.5:3.4), although the ability of Ca2+, Sr2+ and Ba2+ to shift its voltage dependence remained the same as for the fast channel.

Single-Cell RT-PCR and Functional Characterization of Ca2+Channels in Motoneurons of the Rat Facial Nucleus

Voltage-dependent Ca2+ channels are a major pathway for Ca2+ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltage-gated Ca2+ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and approximately 5% of L-type. Despite the dominating role of P-type Ca2+ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca2+ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons. Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the alpha1-subunit, we identified a highly heterogeneous expression pattern of Ca2+ channel alpha1-subunit mRNA in individual neurons consistent with the Ca2+ current components found in the cell bodies and axon terminals. We detected mRNA for alpha1A in 86% of neurons, alpha1B in 59%, alpha1C in 18%, alpha1D in 18%, and alpha1E in 59%. Either alpha1A or alpha1B mRNAs (or both) were present in all neurons, together with various other alpha1-subunit mRNAs. The most frequently occurring combination was alpha1A with alpha1B and alpha1E. Taken together, these results demonstrate that the Ca2+ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.

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).

Developmental changes in the expression of low-voltage-activated Ca2+ channels in rat visual cortical neurones.

1. The functional properties of low-voltage-activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole-cell patch-clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12). 2. It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I-II) and deep (V-VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III-IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 microM) and were insensitive to Ni2+ (25 microM). 3. The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high-voltage-activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F- applied through the patch pipette were observed. 4. The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin-Huxley equation with an inactivation time constant of 24 +/- 6 ms. The steady-state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half-inactivation, -86.9 mV; steepness coefficient,3.4 mV. 5. It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another ('slow') type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cells is different.

Expression of Ca 2+ channels from rat brain with model phenylketonuria in Xenopus oocytes

Ca 2+ channels expressed in Xenopus oocytes by means of mRNA purified from the brain of the rats subjected to chronic treatment with l-phenylalanine in order to model conditions typical for the congenital disease called phenylketonuria (PKU) were studied using double microelectrode technique. The amplitude of Ca 2+ channel currents ( I Ba, 40 mM Ba 2+ as a charge carrier) directed in the oocytes by mRNA from the brain of the animals with model PKU was significantly smaller compared to the control animals (145±23 nA vs. 270±38 nA, p<0.025) while the voltage-dependence of both currents was similar and typical for that of high voltage-activated (HVA) Ca 2+ channels. No evidence for the expression of low voltage-activated Ca 2+ channels were found. The decrease of the overall HVA Ba 2+ current under model PKU occurred primarily at the expense of the decaying, ω-conotoxin-sensitive component which accounted for about 64% of the total current amplitude in control, and apparently was associated with the activity of the expressed N-type Ca 2+ channels. ω-Aga-IVA-sensitive, P/Q component of I Ba that contributed not more than 10% to the total current in control showed no change under PKU conditions. In addition to the decreased amplitude, Ba 2+ current from model PKU animals showed accelerated run-down during prolonged recording (50%/h compared to 15%/h in control). Our data suggest that hyperphenylalaninemic conditions affect the expression of preferentially N-type Ca 2+ channels via the reduction of their specific mRNA content as well as influence the type and manner of channels regulation. The underexpression of N-type Ca 2+ channels is consistent with the decrease in the overall number of synaptic contacts during PKU and may be one of the factors contributing to the severe damage of the brain function.

Octopamine modulates ionic currents and spiking in dorsal unpaired median (DUM) neurons.

Modulatory effects of octopamine on ionic currents and spiking in isolated cockroach dorsal unpaired median neurons were investigated by means of the chopped voltage-/current-clamp and the patch-clamp technique. Octopamine increased the spiking frequency at concentrations < or = 10 microM and reduced it at > 10 microM. It enhanced a low voltage-activated Ca2+ current at 1 to 100 microM. At concentrations up to 10 microM two components of Ca2+-activated K+ current were potentiated. At > 10 microM octopamine in addition reduced a high voltage-activated Ca2+ current and the Ca2+-activated K+ current. A membrane permeant cAMP-analogue imitated the effects obtained at high octopamine concentration. Octopamine and cAMP in addition reduced a depolarizing resting current at both low and high concentration. Possible mechanisms and physiological significance are discussed for the opposite effects of octopamine observed at low versus high concentrations.