Low-voltage Activity Research Articles

Single-channel properties of four calcium channel types in rat motoneurons

Previous studies have demonstrated multiple components of whole-cell calcium currents in hypoglossal motoneurons (HMs); HMs possess a low-voltage-activated (LVA) current and three types of high-voltage-activated (HVA) calcium currents based on sensitivity to omega-Aga IVA, omega-Conotoxin GVIA (omega-CgTx) and dihydropyridine analogs (DHPs). In the present study, we recorded single-calcium channel activities from HMs using a cell-attached patch-clamp method and found four types of channels that could be discriminated based on kinetics, voltage dependency, DHP sensitivity, and single-channel conductances. The average single-channel conductances with 110 mM barium as a charge carrier were 7, 14, 20, and 28 pS. T-type channels had a single-channel conductance of 7 pS, activated at the most negative potentials for the calcium channels and inactivated during depolarization. L-type channels (DHP-sensitive channels) did not inactivate during depolarization and had a 28-pS single-channel conductance. Based on kinetics and sensitivity to holding potential, it is likely that the channels with conductances of 14 pS and 20 pS represent N-type and P-type channels, respectively. The N-type channel (14 pS) was sensitive to holding potential, showed modal gating, and inactivated during maintained depolarizations, whereas the P-type channel (20 pS) was rather insensitive to holding potential and did not inactivate during depolarization.

Characterization of Ca2+ channel currents in cultured rat cerebellar granule neurones.

1. High-threshold voltage-gated calcium channel currents (IBa) were studied in cultured rat cerebellar granule neurones using the whole-cell patch clamp technique with 10 mM Ba2+ as the charge carrier. The putative P-type component of whole-cell current was characterized by utilizing the toxin omega-agatoxin IVA (omega-Aga IVA) in combination with other blockers. 2. omega-Aga IVA (100 nM) inhibited the high voltage-activated (HVA) IBa by 40.9 +/- 3.4% (n = 27), and the dissociation constant Kd was 2.7 nM. Maximal inhibition occurred within a 2-3 min time course, and was irreversible. The isolated omega-Aga IVA-sensitive current was non-inactivating. 3. omega-Aga IVA exhibited overlapping selectivity with both N- and L-channel blockers; omega-conotoxin GVIA (omega-CTX GVIA) (1 microM) and the dihydropyridine (-)-202-709 (1 microM), respectively. Together these toxins reduced the omega-Aga IVA-sensitive component to just 4.5 +/- 1.4% (n = 3). Thus only a small proportion of the current can be unequivocally attributed to P-type current. Inhibition of the HVA IBa by omega-Aga IA also reduced the proportion of omega-Aga IVA-sensitive current to 28.0 +/- 3.2% (n = 3). 4. Application of omega-Aga IVA and a synthetic form of funnel-web toxin, N-(7-amino-4-azaheptyl)-L-argininamide (sFTX-3.3; 10 microM), produced an additive block of the HVA IBa. Consequently these two toxins do not act on the same channel in cerebellar granule neurones. 5. omega-Aga IVA inhibition of low voltage-activated (LVA) IBa was studied in the ND7-23 neuronal cell line. omega-Aga IVA (100 nM) reduced the LVA current by 41.3 +/- 3.2% (n = 17) in a fully reversible manner with no shift in the steady-state inactivation of the channel. 6. A component of current insensitive to N-, L- and P-channel blockers remained unclassified in all our studies. This component, and also that remaining following block by omega-Aga IVA and omega-Aga IA, exhibited relatively rapid, although incomplete, inactivation compared to the other currents isolated in this study. 7. In conclusion, omega-Aga IVA inhibits a component of current in cultured cerebellar granule neurones which overlaps almost completely with that inhibited by L- and N-channel blockers. In addition, a large component of whole-cell current in these neurones still remains unclassified.

Differential beta‐adrenergic regulation and phenotypic modulation of voltage‐gated calcium currents in rat aortic myocytes.

1. We studied the beta-adrenergic regulation of voltage-gated Ca2+ channel currents using the whole-cell patch-clamp technique (18-22 degrees C) in freshly isolated and in cultured (1-20 days) rat aortic vascular smooth muscle cells (VSMCs). These currents include a transient low-voltage-activated (LVA) current and two L-type-related high-voltage-activated currents (HVA1 and HVA2, respectively). 2. At 10 microM, the beta-adrenergic agonist, isoprenaline, increased the HVA2 current (65 +/- 30%, n = 10) but had no effect on LVA and HVA1 currents. This potentiation was dose dependent in the range 0.01-10 microM, developed with a slow time course and was mimicked by elevating intracellular cyclic AMP using the permeant analogue dibutyryl cyclic AMP (100 microM). 3. In the well-differentiated freshly isolated myocytes, only the HVA1 current was recorded. In cultured cells, a predominant frequency of occurrence of LVA and HVA1 currents was observed in modulated and differentiated myocytes, respectively. The occurrence of the HVA2 current was stable during culture but this current disappeared when the cells were confluent. It was retrieved when the confluent cells were dispersed and subcultured. 4. In conclusion, we present evidence for a differential beta-adrenergic regulation of three types of Ca2+ channel current in adult rat aortic VSMCs. The differential expression of these currents, associated with marked changes in cell phenotypes in vitro, suggests that they serve distinct physiological functions.

Properties and function of low- and high-voltage-activated Ca2+ channels in hypoglossal motoneurons

Calcium influx through voltage-gated Ca2+ channels plays an important role in neuronal function. In a thin-slice preparation of neonatal rat hypoglossal motoneurons (HMs) we recorded Ba2+ currents through voltage-gated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that HMs have low-voltage-activated (LVA) and at least three types of high-voltage-activated (HVA) Ca2+ channels (omega-Aga-IVA sensitive, omega CgTx sensitive, and dihydropyridine sensitive), based on pharmacological and voltage-dependent properties. Of the Ca2+ current activated at 0 mV from a holding potential of -70 mV, approximately one-half was omega-Aga-IVA (200 nM) sensitive, one-third was omega-CgTx (3 microM) sensitive, whereas only 6% was DHP (nimodipine; 10 microM) sensitive. The residual current, after applying these three antagonists, had characteristics of LVA Ca2+ currents. Based on this pharmacology we found that Ca2+ entry during a single action potential (AP) through LVA Ca2+ channels has a different role from CA2+ entry through HVA Ca2+ channels. Ca2+ influx through omega-Aga-IVA-sensitive and omega-CgTx-sensitive HVA Ca2+ channels activates Ca(2+)-activated K+ channels responsible for the AP afterhyperpolarization. On the other hand, Ca2+ entry through LVA Ca2+ channels is responsible for spike afterdepolarization and provides Ca2+ for the Ca(2+)-activated K+ channels that contribute to AP repolarization.

New dihydropyridine lacidipine persistently inhibits L-type calcium currents in GH3 cells.

We studied the effects of two antagonist dihydropyridines (DHPs) on calcium currents in cultured pituitary GH3 cells by the perforated patch-clamp method. At depolarizing voltages, GH3 cells present both the low-voltage-activated (LVA), fast-inactivating T-type calcium channel and the high-voltage-activated (HVA), fast-deactivating L-type calcium channel. As already demonstrated in whole-cell experiments and in perforated-patch configuration, 1 microM nimodipine reversibly inhibited < or = 80% of L-type calcium channels when applied in the external bath solution. At concentrations of 0.1-1 microM, the new DHP lacidipine inhibited L-type calcium current, but this inhibition was very persistent and never reversed, even after prolonged washout, in a typical perforated patch-clamp experiment (< or = 2h). Like that of other DHPs, the potency of lacidipine block increases at more depolarizing holding potentials (HPs). The time needed to inhibit 50% (t1/2) of L-type calcium current was decreased by increasing lacidipine concentration; t1/2 was 22 +/- 3 s with addition of 1 microM lacidipine; this concentration inhibited < or = 86 +/- 1% of the L-type current. The persistent blocking induced by lacidipine can be explained in terms of a strong interaction of the drug with the membrane phospholipids, as a consequence of the enhanced hydrophobicity and specific location of this molecule with respect to other DHPs.

Local anesthetics depress the calcium current of rat sensory neurons in culture.

Local anesthetics are known to inhibit the voltage-gated sodium current (INa) of the nerve membrane, but it has not been fully studied whether anesthetic concentrations of local anesthetics depress the voltage-gated calcium current (ICa) of mammalian neurons. The effects of local anesthetics on ICa evoked in cultured rat dorsal root ganglion cells were studied. Whole cell patch clamp recordings were made from rat dorsal root ganglion cells cultured for 1-3 weeks. ICa was recorded using patch electrodes filled with Cs-aspartate in Na(+)-free external solution containing 5 mM-Ba2+. All drugs, including local anesthetics, were applied by miniperfusion from micropipettes by pressure ejection. Tetracaine (300 microM) depressed the peak amplitudes of high voltage-activated (HVA)-ICa to 22.6 +/- 8.8% of control values (n = 14) without affecting the current-voltage relation. A tetracaine dose-response curve for HVA-ICa indicated an apparent dissociation constant of 79.5 microM. Tetracaine (30 microM) depressed nicardipine-sensitive HVA-I(Ca) (L-type) to 14.3 +/- 6.7% (n = 6), omega-conotoxin-sensitive HVA-ICa (N-type) to 81.6 +/- 9.6% (n = 7), and low voltage-activated (LVA)-ICa (T-type) to 65.1 +/- 11.1% (n = 6) of their respective controls. Local anesthetics other than tetracaine also depressed HVA-ICa but were of different potency; the rank sequence was dibucaine > tetracaine > bupivacaine >> procaine = lidocaine. These results suggest that both HVA-ICa and LVA-ICa are depressed by tetracaine used at the concentrations required for spinal anesthesia and that the L-type Ca2+ channel among Ca2+ channel subtypes is the most susceptible to tetracaine. A good correlation between local anesthetic potencies to inhibit HVA-ICa and their anesthetic potencies implies that the inhibition of calcium influx through voltage-gated channels may contribute to spinal anesthetic mechanisms.

Whole-cell and single-channel calcium currents in guinea pig basal forebrain neurons.

1. Whole-cell and single-channel patch-clamp recordings of calcium (Ca2+) currents were made in acutely dissociated neurons from the medial septum (MS) and nucleus of the diagonal band (nDB) of adult guinea pig. Barium (Ba2+) was used as the charge carrier across the Ca2+ channel and multiple channel types were identified in different cell types. 2. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) currents were distinguished on the basis of steady-state voltage dependence, activation and inactivation properties, and pharmacological sensitivity. HVA currents had activation thresholds approximately 20 mV more positive than LVA currents. Steady-state inactivation of HVA currents was approximately 50% when the holding potential was shifted from -80 to -40 mV. 3. The dihydropyridines had consistent effects on HVA currents. The amplitude was increased and the activation threshold shifted by 10 mV in the hyperpolarizing direction in the presence of the agonist Bay K 8644 (2-5 microM). The antagonist nifedipine (10 microM) produced approximately 50% inhibition of HVA currents from a holding potential of -80 mV. 4. A second component of the HVA current was blocked by omega-conotoxin (omega-CTX) (300-700 nM). At a holding potential of -80 mV, omega-CTX inhibited 45% of the HVA current. 5. LVA currents were activated near -70 mV and displayed time-dependent inactivation during a 200- to 300-ms voltage step. Voltage-dependent inactivation of LVA currents was also observed and could be described by a single Boltzman relationship with a half-inactivation potential of -84 mV. LVA currents were not significantly changed by Bay K 8644 and were not blocked by low concentrations of nifedipine or omega-CTX. 6. Single voltage-gated Ca2+ channels were investigated using cell-attached patches. In these experiments, 100 mM Ba2+ was used in the patch pipette and the membrane potential was zeroed with isotonic potassium (K+)-aspartate. A low-conductance channel was activated at negative potentials and inactivated rapidly during a 200- to 300-ms voltage step. Unitary amplitudes were determined at different membrane potentials with single-channel conductances calculated to be 7.8 +/- 1.2 (SD) pS. These channels were not blocked by nifedipine (10 microM) and appeared similar to T channels previously reported in both peripheral and central neurons. Ensemble averages from cell-attached patches of T channels resembled LVA currents recorded in the whole-cell configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

The general anesthetic propofol inhibits transmembrane calcium current in chick sensory neurons.

The action of propofol on voltage-gated calcium channels was investigated in cultured dorsal root ganglion neurons from chick embryos. The Ca2+ current was measured by using the patch-clamp technique in whole cell configuration. Low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents were selected by means of appropriate stimulation protocols. Propofol (0.3 mM) inhibited the LVA T-type current by 80% (P < 0.001). The same concentration of propofol reduced the HVA Ca2+ current with a high variability (10%-75%). The inactivation time constant of the HVA current was also shortened to 50% by propofol. omega-Conotoxin and nifedipine were used to discriminate between the HVA N- and L-type current components. Only the L-type component was strongly depressed (75%) by propofol (P < 0.001); different effects on the HVA current might, therefore, reflect different percentages of L- and N-type channels in neurons. We conclude that propofol inhibits the T-type and L-type components of the Ca2+ current. This inhibition may play a role in the cardiovascular side effects clinically observed.

Mechanosensitivity of voltage-gated calcium currents in rat anterior pituitary cells.

Sensitivity of voltage-activated calcium currents to flow-induced mechanical stress was examined in enriched populations of rat anterior pituitary somatotrophs. Voltage-activated calcium currents were recorded with the whole-cell configuration of the patch-clamp technique. Pituitary cells were exposed to flow (from pipettes) which was produced by a hydrostatic pressure of about 3 cmH2O. In 92% of the cells studied (n = 87 cells) flow reduced the amplitude of both low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents. These effects of flow on calcium currents did not result from changes in either seal resistance or leak conductance of the cell and were dependent on the magnitude of flow. The effect of flow is selective. We found that LVA calcium currents were substantially more sensitive to flow than HVA calcium currents. Under constant flow conditions, LVA calcium currents were reduced by 57.6 +/- 29.6% (S.D.), whereas HVA currents (recorded from the same cells) were reduced by only 17.8 +/- 15.9% (S.D.). The effects of flow on calcium currents were associated with effects on their related calcium tail currents. Slowly deactivating calcium tail currents were reduced by 75.3 +/- 25.6% (S.D.), whereas rapidly deactivating calcium tail currents were reduced by 29.1 +/- 14.4% (S.D.). The effect of flow on calcium currents was not associated with any significant shift in the activation curves of the calcium currents (voltage range -60 to +30 mV), suggesting that the effect of flow is not voltage dependent. The effect of flow is not dependent on activation of calcium currents during the exposure to flow. Calcium currents which were evoked immediately after cessation of the exposure to flow were reduced in amplitude and recovered to control values. Possible mechanisms underlying the flow effect and possible physiological relevance of the effect on pituitary cells are discussed.

Effect of mild hypocapnia on fetal breathing and behavior in unanesthetized normoxic fetal lambs.

We hypothesized that the level of arterial PCO2 (PaCO2) affects the incidence of fetal breathing movements and electrocorticographic (ECoG) states in chronically instrumented fetal sheep. Six fetuses of 128-132 days gestational age were instrumented for recording fetal behavior and for later connection to an extracorporeal membrane oxygenation (ECMO) system to change fetal blood gases. Before ECMO fetal arterial pH and blood gases were pH 7.40 +/- 0.01, PaCO2 42.9 +/- 1.5 Torr, and arterial PO2 (PaCO2) 19.2 +/- 1.7 Torr; during ECMO in normocapnia they were pH 7.37 +/- 0.01, PaCO2 46.1 +/- 0.7 Torr, and PaCO2 27.6 +/- 3.0 Torr; and during ECMO in mild hypocapnia they were pH 7.47 +/- 0.01, PaCO2 35.3 +/- 1.7 Torr, and PaCO2 26.6 +/- 1.7 Torr. The overall incidence of breathing movements, the incidence of breathing movements during low-voltage (LV) ECoG activity, and the mean duration of periods of breathing decreased significantly during hypocapnia. Fetal ECoG activity showed normal cycling during the periods of mild hypocapnia, and the mean duration of LV ECoG periods did not change. During mild hypocapnia, eye movements remained associated with LV ECoG activity and nuchal electromyographic activity remained associated with high-voltage ECoG activity. These results suggest that the presence of breathing movements in fetal life is not only dependent on the behavioral state but also on the level of fetal PaCO2.

Single-channel properties of high- and low-voltage-activated calcium channels in rat pituitary melanotropic cells.

1. Single-channel properties of voltage-dependent calcium channels were investigated in rat melanotropes in short-term primary culture. Unitary currents were resolved using the cell-attached configuration. 2. Depolarizations higher than -50 mV activated a population of 8.1-pS calcium channels [low-voltage activated (LVA)]. The LVA channel ensembles displayed a monoexponential time course of inactivation and a sigmoidal time course of activation fitted best by an m2h Hodgkin-Huxley-type model. Microscopic kinetic analysis suggested that at least one open state, two closed states, and one inactivated state are involved in channel gating. 3. At potentials positive to -20 mV a second class of calcium channels was activated with a conductance of 24.7 pS [high-voltage activated (HVA)]. HVA channels display different gating modes. Gating with high open probability (mode 2) and low open probability (mode 1) as well as blank traces (mode 0) are observed. The HVA channels were heterogeneous with respect to their inactivation properties. Ensembles that decayed entirely during a 300-ms test pulse as well as nondecaying ensembles were observed. Both HVA channel subtypes displayed sigmoidal activation, which was fitted by an m2 model. Microscopic kinetic analysis suggested that at least one open state and two closed states are involved in mode two gating of both HVA channel subtypes. 4. Depolarizing prepulses did not recruit or facilitate calcium channel activity in response to a test pulse, but inactivating HVA channel activity was strongly reduced. Depolarizing prepulses (+50 mV) did not affect the probability of opening of the noninactivating HVA channel. 5. The voltage dependence and kinetics of the LVA as well as both HVA channels are in good agreement with previously published data on the properties of the various calcium current components derived from whole-cell recordings of rat melanotropes. The data suggest that a T-type as well as two L-type channels (an inactivating and noninactivating channel) underlie the calcium current in these cells.

Induction of a low voltage-activated, fast-inactivating Ca2+ channel in cultured bone marrow stromal cells by dexamethasone.

The production of biochemical markers associated with the osteoblastic phenotype, and accompanying changes in the expression of voltage-operated Ca2+ channels, have been examined in rat bone marrow stromal cell cultures treated with dexamethasone (10(-8) M). Whole cell clamp analysis of voltage-operated Ca2+ channels in control cultures (using Ba2+ as the charge carrier) revealed primarily a high voltage-activated (HVA), slowly inactivating current, which was enhanced two- to threefold by treatment of the cells with Bay K 8644 (300 nM) and inhibited by nifedipine (4 microM). In dexamethasone-treated cultures, the I-V relationship for inward current was shifted to more positive potentials in comparison with control cells. Most cells in these cultures possessed both the HVA current and also a faster inactivating, low-voltage-activated (LVA), nifedipine-resistant current. These two currents could be separated both by nifedipine and by the use of steady state inactivation of the LVA current. The two components of the Ba2+ current varied widely in their relative size. The combination of LVA and HVA currents seen in dex-induced stromal cells resembles records of voltage-operated Ca2+ channels from cultures of calvarial osteoblasts.

Low-threshold Ca2+ current and its role in spontaneous elevations of intracellular Ca2+ in developing Xenopus neurons [published erratum appears in J Neurosci 1994 Mar;14(3):following table of contents

Amphibian spinal neurons exhibit spontaneous elevations of intracellular calcium at early stages of development. The underlying calcium influx involves high-voltage-activated (HVA) currents. To begin to understand how they are triggered, we have studied the biophysical properties and developmental function of low-voltage-activated (LVA) T-type calcium current of neurons cultured from the embryonic neural plate. T current was recorded from young neurons (6-9 hr in vitro) and from mature neurons (18-48 hr in vitro) using whole-cell voltage clamp. For both young and mature neurons, T current has a low threshold and is activated at membrane potentials positive to -60 mV in 2 mM extracellular calcium. The current is maximal at -35 mV with a mean peak amplitude of approximately 50 pA. Nickel blocks both LVA and HVA currents, but the former are 20-fold more sensitive. Amiloride also blocks T current selectively. T current is recorded in 87% of young neurons. This percentage drops to 67% in mature neurons after 1 d in culture and to 35% in mature neurons after 2 d in culture. There are no significant developmental changes in T current threshold, peak density, time course of activation and inactivation, and pharmacological sensitivity to blockers from 6 to 48 hr in culture. Spontaneous transient calcium elevations in young neurons assayed by fluo-3 fluorescence are blocked by nickel or amiloride at concentrations that specifically block T current. T current has the lowest threshold among other inward currents in young neurons. Moreover, mathematical simulations show that T current lowers the threshold of the action potential by 15 mV. We conclude that T current can depolarize cells and trigger action potentials, and constitutes part of the cascade of events leading to spontaneous elevations of intracellular calcium in cultured neurons at early stages of differentiation.

Calcium conductances and their role in the firing behavior of neonatal rat hypoglossal motoneurons

1. The role of calcium conductances in action potential generation and repetitive firing behavior of hypoglossal motoneurons (HMs) was investigated using intracellular recording and patch-clamp techniques in a brain stem slice preparation of neonatal rats (0-15 days old). 2. The action potential was followed by an afterdepolarization (ADP). The ADP was voltage dependent, increasing with membrane hyperpolarization. Raising the extracellular Ca2+ concentration or replacing Ca2+ with Ba2+ increased the ADP amplitude, whereas replacement of Ca2+ with Mn2+ blocked it. The ADP was partially reduced by amiloride and low concentrations of Ni2+. 3. The firing behavior of individual neonatal HMs was influenced by membrane potential. From depolarized potentials, HMs fired tonically in response to a depolarizing current pulse, whereas from more hyperpolarized membrane potentials (more negative than -70 mV), a subset of HMs fired an initial burst of action potentials followed by a prolonged afterhyperpolarization and tonic firing. The incidence of burst-firing behavior was highest among young motoneurons and disappeared by the tenth postnatal day. In addition, prominent rebound depolarizations characterized the response of neonatal motoneurons to hyperpolarizing prepulses. 4. Pharmacological characterization of the rebound depolarization demonstrated that it was calcium dependent. Its amplitude was insensitive to tetrodotoxin and it was eliminated by replacement of Ca2+ with Mn2+ or addition of Ni2+. Amiloride (1-1.5 mM) had no effect on the rebound response or burst firing. 5. The presence of high-threshold calcium spikes was detected at all postnatal ages, but only after blockade of outward currents with intra- or extracellular tetraethylammonium. The high-threshold calcium spikes were greatly enhanced when Ba2+ replaced Ca2+. 6. Calcium currents of neonatal HMs were characterized in whole-cell patch-clamp recordings of thin medullary slices under conditions that minimized voltage-dependent Na+ and K+ currents. Low voltage-activated (LVA) and a high voltage-activated (HVA) calcium current components were identified on the basis of their voltage thresholds for activation, kinetics of inactivation, and pharmacological sensitivity. 7. The LVA calcium current began to activate at around -60 mV and inactivated nearly completely within 100 ms. Complete steady-state inactivation occurred at potentials more positive than -60 mV. The LVA current was selectively reduced by 1 mM amiloride (31%). 8. A larger-amplitude calcium current activated at potentials around -35 mV. Inactivation of this HVA current was slower than that of the LVA current and incomplete. About 1/3 of this current was sensitive to 1 microM omega-conotoxin GVIA, whereas a smaller fraction was blocked by 10 microM nifedipine.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen increases low voltage-activated calcium current density in GH3 anterior pituitary cells.

The effects of estrogen (17 beta-estradiol) on calcium current density were examined in the GH3 anterior pituitary cell line. The addition of 1 nM estrogen to the growth medium induces a 4- to 5-fold increase in low voltage-activated (LVA) Ca2+ current density without affecting high voltage-activated Ca2+ current density. The increase is significant after 24 h, reaches a maximum level in 3 days, and reverses with a similar time course when the estrogen is withdrawn. The EC50 for estrogen stimulation of LVA Ca2+ current density is about 30 pM. The LVA current induced by estrogen has similar voltage dependence and time course of activation, inactivation, and deactivation as controls. The effect of estrogen requires protein synthesis, since it is blocked by cycloheximide. It is suggested that the estrogen-induced increase in LVA Ca2+ current density may be due to an increase in the number of functional channels in the membrane. This effect could be due to insertion of new channels or synthesis of a protein that converts preexisting silent channels into functional channels.

Development of calcium currents in cultures of mouse spinal cord and dorsal root ganglion neurones.

Cultured spinal cord (SC) and dorsal root ganglion (DRG) neurones of 11-13 day old foetal mice were investigated electro-physiologically during differentiation in vitro using the whole-cell patch-clamp technique. High-voltage-activated calcium currents (HVA) and low-voltage-activated (LVA) calcium currents were measured using barium ions as charge carrier. During differentiation in vitro the soma diameter of SC-neurones increased with age (0-42 days in vitro) from 10.3 +/- 2.7 microns to 25.1 +/- 5.9 microns. The capacitance of the soma increased from 7.4 +/- 2.3 pF to 34 +/- 6 pF. The inward calcium current amplitudes increased from 200 pA to 3 nA, while the LVA current amplitude increased only from 50 pA to 100-150 pA. The currents per membrane area through HVA calcium channels increased in the investigated time while the currents through LVA channels decreased.

The whole-cell calcium current in acutely dissociated magnocellular cholinergic basal forebrain neurones of the rat.

1. The electrophysiological and pharmacological characteristics of the calcium current (ICa) in acutely dissociated magnocellular cholinergic basal forebrain neurones from 11- to 14-day-old post-natal rats were studied using the whole-cell patch-clamp technique. 2. All cells exhibited a small transient low-voltage-activated (LVA) current with half-activation and half-inactivation potentials of -40.2 and -49.3 mV and slope factors for activation and inactivation of 4.82 and 3.85 mV per e-fold change in membrane potential (Vm) respectively. Activation and inactivation rates for the LVA current were highly voltage dependent. For test potential changes from -50 to -20 mV, the time-to-peak of the current decreased from 39.1 to 6.4 ms, and the time constant of current decay decreased from 81.7 to 15.5 ms. 3. A high-voltage-activated (HVA) component of ICa could be elicited at threshold voltages between -46 and -30 mV from a holding potential (VH) of -80 mV. The HVA current peaked around 0 mV; a 10-fold increase in [Ca2+]o produced a 13 mV positive shift in the peak, whilst the amplitude of the current showed an approximately hyperbolic relationship to [Ca2+]o with half-saturation at 2.5 mM. The transient phase of the HVA current could be described by two exponential functions with time constants tau fast and tau slow of 16.2 and 301 ms. Steady-state inactivation of the transient and extrapolated true sustained (pedestal) components of HVA current were described by Boltzmann equations, with half-inactivation potentials (slope factors) of -47.3 mV, (9.04) and -29.2 mV (11.8) respectively. 4. omega-Conotoxin (omega-CgTX; 100 nM) irreversibly inhibited a kinetically distinct component of HVA current but had no effect upon the transient LVA current. The omega-CgTX-sensitive current could not be distinguished from the control HVA current by the voltage dependence of its activation or inactivation rates. 5. Low concentrations of amiloride (< or = 300 microM) or Ni2+ (< or = 5 microM) selectively inhibited the transient LVA current, with IC50 values of 97 and 5 microM respectively. Cd2+ (< or = 1 microM) selectively blocked a component of HVA current. At higher concentrations, Cd2+ and Ni2+ were non-selective and totally blocked all components of ICa. 6. The lanthanide ions Gd3+ and La3+ produced saturable incomplete block of the HVA current. Maximally effective concentrations of Gd3+ (100 microM) or La3+ (30 microM) inhibited 76.5 and 41.2% respectively of the sustained component of HVA current with corresponding IC50 values of 2.2 and 1.1 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations in membrane potential after axotomy at different distances from the soma of an identified neuron and the effect of depolarization on neurite outgrowth and calcium channel expression.

1. Intracellular recordings were made from the soma of an identified neuron B5 within the buccal ganglion of the mollusc, Helisoma trivolvis, during axotomy induced by crushing or cutting the esophageal nerve. Axotomy was associated with a rapid depolarization and occasionally a burst of action potentials (injury discharge). The magnitude of the membrane depolarization in the soma in response to axotomy decayed exponentially when the distance between the soma and site of injury was increased. Input resistance measurements taken during axotomy showed that a barrier to current flow formed rapidly and gradually recovered within 2 h. A barrier to the diffusion of intracellularly injected carboxyfluorescein formed at the site of injury within 15 min of axotomy. 2. To examine the effect of chronic depolarization on neurite outgrowth, the extracellular potassium ion concentration [K+]o was manipulated. The membrane potential of neurons B5 exhibited a 51.8 mV/decade potassium dependence between 20 and 150 mM [K+]o. The initiation of neurite outgrowth from axons crushed 800 microns from the soma and bathed in different concentrations of [K+]o was examined by fluorescence microscopy after filling neurons with Lucifer yellow. We compared the percentage of axons with sprouts 9 and 24 h after organ culture in saline containing [K+]o ranging from 0.1 to 50 mM. Sprouting occurred from 33% of neurons B5 in normal saline (1.7 mM [K+]o) after 9 h and from 100% of neurons after 24 h. No sprouting was observed from neurons B5 9 or 24 h after axotomy when bathed in saline containing reduced or elevated concentrations of [K+]o. 3. To examine the effects of chronic depolarizatin on neurite outgrowth over several days, neurons B5 were axotomized close to the soma and maintained in organ culture in Liebovitz medium (defined medium or medium conditioned with central ganglia). Neurite outgrowth was ranked from 0 to 5 after filling neurons with Lucifer yellow, and our analysis indicated that a small increase in neurite outgrowth occurred in medium supplemented with 10 mM potassium. 4. Elevated potassium did not trigger neurite outgrowth from isolated neurons B5 in cell culture within defined medium, but whole-cell patch-clamp analysis revealed that chronic depolarization associated with elevated potassium altered the expression of calcium currents. Low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium currents were detected in acutely isolated neurons B5.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.

Kinetic and pharmacological properties of low voltage-activated Ca2+ current in rat clonal (GH3) pituitary cells.

1. Low voltage-activated (LVA) Ca2+ current in clonal (GH3) pituitary cells was studied with the use of the whole-cell recording technique. The use of internal fluoride to facilitate the rundown of high voltage-activated (HVA) Ca2+ current allowed the study of LVA current in virtual isolation. 2. In 10 mM [Ca2+]o, detectable LVA current begins to appear at about -50 mV, with half-maximal activation occurring at -33 mV. The time course of activation was best described by a Hodgkin-Huxley expression with n = 3, suggesting that at least three closed states must be traversed before channel opening. 3. Deactivation was found to vary exponentially with membrane potential between -60 and -160 mV, indicating that channel closing is rate-limited by a single, voltage-dependent transition. 4. Onset and removal of inactivation between -40 and -130 mV were best described by the sum of two exponentials. Between -80 and -130 mV, both components of removal of inactivation showed little voltage dependence, with time constants of approximately 200-300 ms and 1-2 s. At membrane potentials above -40 mV, a single component of inactivation onset was detected. This component was voltage independent between -20 and +20 mV (tau = 22 ms). Thus inactivation of LVA current is best described by multiple, voltage-in-dependent processes. 5. Significant inactivation of LVA current occurred at -65 mV without detectable macroscopic current. This suggests that inactivation is not strictly coupled to channel opening. 6. Peak LVA current increased with increasing [Ca2+]o, with saturation approximately 50 mM. The Ca(2+)-dependence of peak LVA current was reasonably well described by a single-site binding isotherm with half-maximal LVA current at approximately 7 mM. 7. LVA current in GH3 cells was largely resistant to blockade by Ni2+. The relative potency of inorganic cations in blocking GH3 LVA current was (concentrations which produced 50% block): La3+ (2.4 microM) greater than Cd2+ (188 microM) greater than Ni2+ (777 microM). 8. Several organic agents, including putative LVA blockers, HVA current blockers and various anesthetic agents, were tested for their ability to block LVA current. The concentrations that produced 50% block are as follows: nifedipine (approximately 50 microM), D600 (51 microM), diltiazem (131 microM), octanol (244 microM), pentobarbital (985 microM), methoxyflurane (1.41 mM), and amiloride (1.55 mM). Phenytoin and ethosuximide produced 36 and 10% block at 100 microM and 2.5 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)