Low Voltage-activated Currents Research Articles

Elevated thalamic low-voltage-activated currents precede the onset of absence epilepsy in the SNAP25-deficient mouse mutant coloboma.

Recessive mutations in genes encoding voltage-gated Ca2+ channel subunits alter high-voltage-activated (HVA) calcium currents, impair neurotransmitter release, and stimulate thalamic low-voltage-activated (LVA) currents that contribute to a cortical spike-wave epilepsy phenotype in mice. We now report thalamic LVA current elevations in a non-Ca2+ channel mutant. EEG analysis of Coloboma (Cm/+), an autosomal dominant mutant mouse lacking one copy of the gene for a synaptosomal-associated protein (SNAP25) that interacts with HVA channels, reveals abnormal spike-wave discharges (SWDs) in the behaving animal. We compared the biophysical properties of both LVA and HVA currents in Cm/+ and wild-type thalamic neurons and observed a 54% increase in peak current density of LVA currents evoked at -50 mV from -110 mV in Cm/+ before the developmental onset of seizures relative to control. The midpoint voltage for steady-state inactivation of LVA currents in Cm/+ was shifted in a depolarized direction by 8 mV before epilepsy onset, and the mean time constant for decay of LVA Ca2+ currents at -50 mV was also prolonged. No significant differences were found in recovery from inactivation of LVA currents or in HVA current densities and kinetics. Our data demonstrate that a non-Ca2+ channel subunit gene mutation leads to potentiated thalamic LVA currents that precede the appearance of SWDs and that altered somatodendritic HVA currents are not required for abnormal thalamocortical oscillations. We suggest that presynaptic release defects shared by these mutants lead to postsynaptic LVA excitability increases in thalamic pacemaker neurons that favor rebound bursting and absence epilepsy.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells.

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 +/- 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 +/- 2.4 and 6.7 +/- 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 +/- 0.97 and 7.5 +/- 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of alpha1G (CaV3.1) and alpha1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Calcium current subtypes in GnRH neurons.

Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% +/- 2%. The remaining HVA calcium current passed through N-type (27% +/- 3%), P-type (15% +/- 1%), Q-type (18% +/- 3%), and R-type (15% +/- 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4-10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.

Enhancement of low-voltage-activated calcium currents by group II metabotropic glutamate receptors in rat retinal ganglion cells

Current through voltage-gated calcium channels of rat retinal ganglion cells was recorded using the whole-cell patch-clamp technique. All cells displayed high-voltage-activated currents, and 75% of these also displayed low-voltage-activated (LVA) currents. Currents could be separated on the basis of their voltage/time dependence and sensitivity to nickel ions. The group II metabotropic glutamate receptor (mGluR) agonist (2 R,4 R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC; 100 μM) increased LVA current by 40% as did the nonselective mGluR agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid (tACPD; 100 μM). Neither the group I mGluR agonist ( S)-3,5-dihydroxyphenylglycine (100 μM) nor 5-hydroxytryptamine (100 μM) enhanced LVA current. In the presence of ( S)-α-methyl-4-carboxyphenylglycine (100 μM), a group I/II mGluR antagonist, the tACPD-induced enhancement of LVA current was blocked. The voltage dependence of the activation or inactivation kinetics was unchanged in the presence of tACPD. Inclusion in the pipette solution of GDP-β-S (1 mM) blocked the enhancement of the LVA current by APDC, whereas GTP-γ-S (0.5 mM) prevented recovery of the enhancement. The tACPD-mediated enhancement of the LVA current was still present in cells pretreated with pertussis or cholera toxins (500 ng ml −1). Genistein (10 μM) prevented the enhancement of the LVA current. These results suggest that LVA current can be enhanced by activation of mGluR2, by a mechanism that is G-protein dependent and may involve a protein tyrosine kinase step.

Evidence for low-voltage-activated (LVA) calcium currents in the dendrites of tiger salamander retinal ganglion cells.

We have evaluated the spatial distribution of low-voltage-activated calcium currents in ganglion cells of the tiger salamander retina. Whole-cell recordings were obtained from ganglion cells in a retinal slice preparation and from acutely dissociated ganglion cells that were identified through retrograde dye injection. In single dissociated cells, we estimated the magnitude (pA) and current density (pA/pF) of LVA currents in ganglion cells, both with and without dendritic processes. Ganglion cells that retained a portion of their dendritic arbor had larger LVA calcium currents and higher LVA current densities than those which lacked processes. When cell capacitance measurements were used to derive the surface area of the soma and dendritic processes, we concluded that a higher LVA current density was present in the dendrites; we estimate that, on average, the current density in the dendrites is approximately five times that of the soma. The presence of a significant density of LVA calcium channels in the dendrites of ganglion cells suggests that they could be involved in a number of cellular functions, including dendritic integration of synaptic currents, impulse generation, and homeostatic functions related to changes in the intradendritic calcium concentration.

Arachidonic acid-induced inhibition of Ca2+ channel currents in retinal glial (Müller) cells.

Arachidonic acid is a second messenger that has been implicated in several pathological conditions in nervous tissues. The present study was carried out to determine whether the second messenger arachidonic acid modulates currents through voltage-gated Ca2+ channels in freshly isolated Müller glial cells. Whole-cell voltage-clamp recordings were made in human Muller cells to investigate Ba2+ and Na+ currents through high-voltage-activated (HVA) channels, and in rabbit Muller cells to study Na+ currents through low-voltage-activated (LVA) channels. Extracellular application of arachidonic acid reversibly and dose-dependently depressed the amplitude of both LVA (rabbit cells) and HVA currents (human cells). 10 microM arachidonic acid reduced the peak LVA and HVA currents by approximately 70%. A 50% reduction of LVA currents was achieved at 4.7 microM. The block of HVA and LVA currents was not accompanied by alterations in the voltage dependences of current activation and inactivation. A similar reduction of the currents was achieved by 20 microM eicosatetraynoic acid. Since eicosatetraynoic acid mimics the effects of arachidonic acid, it is assumed that arachidonic acid itself rather than its degradation products modulates glial Ca2+ channel activity. This Ca2+ channel inhibition may stabilize Muller cell function during pathological conditions in which arachidonic acid levels are elevated and may participate in the cellular action of neurotransmitters.

Analysis of Ca2+ Currents in Spermatocytes from Mice Lacking Cav2.3 (α1E) Ca2+ Channel

In mammalian male germ-line cells, low-voltage-activated (LVA) Ca2+ current has been identified and its electrophysiological properties have been studied. To investigate whether α12.3 (α1E) subunit of the voltage-dependent Ca2+ channel codes for the LVA current, whole-cell patch clamp and following reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed in pachytene spermatocytes from Cav2.3+/+ and Cav2.3−/− mice. Whole-cell current in acutely dissociated pachytene spermatocytes from Cav2.3+/+ and Cav2.3−/− mice displayed a typical profile of LVA Ca2+ currents and kinetics with no significant differences. Single-cell RT-PCR revealed the expression of Cacna1g in the pachytene spermatocytes from Cav2.3+/+ and Cav2.3−/− mice in which LVA Ca2+ currents were actually recorded. These results suggest that the Cav2.3 channel makes no detectable contribution to the LVA Ca2+ current in the pachytene spermatocyte. Instead, Cav3 family such as Cav3.1 may be the likely candidates responsible for the LVA currents in pachytene spermatocytes.

Calcium channels in Xenopus spinal neurons differ in somas and presynaptic terminals.

Calcium channels play dual roles in cell signaling by promoting membrane depolarization and allowing entry of calcium ions. Patch-clamp recordings of calcium and calcium-dependent currents from the soma of Xenopus spinal neurons indicate key functional differences from those of presynaptic terminals. Both terminals and somas exhibit prominent high-voltage-activated (HVA) calcium current, but only the soma expresses additional low-voltage-activated (LVA) T-type current. Further differences are reflected in the HVA current; N- and R-type channels are predominant in the soma while the terminal calcium current is composed principally of N type with smaller contribution by L- and R-type channels. Potential physiological significance for these different distributions of channel types may lie in the differential channel kinetics. Activation of somatic HVA calcium current occurs more slowly than HVA currents in terminals. Additionally, somatic LVA calcium current activates and deactivates much more slowly than any HVA calcium current. Fast-activating and -deactivating calcium current may be critical to processing the rapid exocytotic response in terminals, whereas slow LVA and HVA calcium currents may play a central role in shaping the somatic firing pattern. In support of different kinetic behavior between these two compartments, we find that somatic calcium current activates a prominent slow chloride current not observed in terminal recordings. This current activates in response to calcium entering through either LVA or HVA channels and likely functions as a modulator of excitability or synaptic input. The restriction of this channel type to the soma lends further support to the idea that differential expression of fast and slow channel types in these neurons is dictated by differences in signaling requirements for somatic and terminal compartments.

Inhibitory effect of 1,1,1-trichloroethane on calcium channels of neurons.

Peripheral neuropathy may remain for some time after 1,1,1-trichloroethane exposure. A variety of Ca2+ channels gives sensory neurons many kinds of transmitting sensory information. We measured calcium currents in sensory neurons from neonatal rat dorsal root ganglion using whole-cell patch-clamp recordings. Trichloroethane reversibly reduced the low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium. The half-inhibitory concentration (IC50) of the HVA and LVA currents was 5.76 x 10(-3) M and 3.99 x 10(-3) M, respectively. The Hill coefficient of the HVA and LVA currents was 0.61 and 1.04, respectively. In assessing voltage dependence for activation and inactivation of calcium currents, only the HVA calcium current was inactivated at greater negative potentials. This may be one of the mechanisms to reduce HVA current. However, activation and inactivation of the LVA currents were not affected by trichlorothane, so inhibition of the LVA currents may have other mechanisms. Calcium currents are thought to be involved in the control of neuronal excitability and neurotransmitter release. The inhibitory effect of trichloroethane on calcium currents may be involved in trichloroethane-induced sensory discomfort.

Developmental regulation of calcium channel-mediated currents in retinal glial (Müller) cells.

Whole cell voltage-clamp recordings of freshly isolated cells were used to study changes in the currents through voltage-gated Ca(2+) channels during the postnatal development of immature radial glial cells into Müller cells of the rabbit retina. Using Ba(2+) or Ca(2+) ions as charge carriers, currents through transient low-voltage-activated (LVA) Ca(2+) channels were recorded in cells from early postnatal stages, with an activation threshold at -60 mV and a peak current at -25 mV. To increase the amplitude of currents through Ca(2+) channels, Na(+) ions were used as the main charge carriers, and currents were recorded in divalent cation-free bath solutions. Currents through transient LVA Ca(2+) channels were found in all radial glial cells from retinae between postnatal days 2 and 37. The currents activated at potentials positive to -80 mV and displayed a maximum at -40 mV. The amplitude of LVA currents increased during the first postnatal week; after postnatal day 6, the amplitude remained virtually constant. The density of LVA currents was highest at early postnatal days (days 2-5: 13 pA/pF) and decreased to a stable, moderate level within the first three postnatal weeks (3 pA/pF). A significant expression of currents through sustained, high-voltage-activated Ca(2+) channels was found after the third postnatal week in approximately 25% of the investigated cells. The early and sole expression of transient currents at high-density may suggest that LVA Ca(2+) channels are involved in early developmental processes of rabbit Müller cells.

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.

Ca 2+ channel-mediated currents in retinal glial (Müller) cells of the toad ( Bufo marinus)

Whole-cell voltage-clamp recordings were used to detect voltage-gated Ca 2+ channels in freshly isolated retinal glial (Müller) cells of the toad ( Bufo marinus). Using Ca 2+ ions (2 mM) as charge carriers (in the presence of 1 mM Mg 2+), no inwardly directed currents could be observed during the application of depolarizing voltage steps. However, after omitting the divalent cations from the bath solution, large-amplitude inwardly directed currents were evoked that were carried by Na + ions, and were mediated by at least two different kinds of Ca 2+ channels, transient low voltage-activated (LVA) channels and sustained high voltage-activated (HVA) channels. While the LVA currents activated at potentials positive to −90 mV and peaked at −40 mV, the HVA currents activated positive to −60 mV and peaked at −20 mV. It is concluded that Müller glial cells of the toad express distinct types of voltage-gated Ca 2+ channels that may be activated, under certain conditions, close to physiological membrane potentials.

LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart.

The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca(2+) currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca(2+) currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca(2+) compared with Ba(2+), although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co(2+) and La(3+) (1 mM) but was particularly sensitive to Ni(2+) ions ( approximately 50% block with 100 microM Ni(2+)) and insensitive to low doses of the dihydropyridine Ca(2+) channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca(2+) currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca(2+) necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca(2+) as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba(2+) for Ca(2+) increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co(2+) and La(3+) ions (1 mM) but was sensitive to micromolar concentrations of nifedipine ( approximately 50% block 10 microM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca(2+) current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca(2+) necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca(2+) currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart.

Developmental changes in low and high voltage-activated calcium currents in acutely isolated mouse vestibular neurons.

1. The development of low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents was studied in neurons acutely dissociated from mouse vestibular ganglia at embryonic stages (E)14, 15, 17 and birth using the whole-cell patch-clamp technique. 2. LVA current was present in almost all neurons tested at stages E14 to E17, although at birth this current was restricted to a few neurons. Two populations of neurons were characterized based on the amplitude of the LVA current. In the first population, LVA current densities decreased between E17 and birth by which time this current tended to disappear in most neurons. A second population of neurons with high density LVA current appeared at E17, and in this group the mean density increased during development. 3. Among HVA currents, the dihydropyridine-sensitive L-type current remained constant between E15 and birth. Over the same period, the density of N- and Q-type currents continuously increased as shown using omega-conotoxin-GVIA (N-type), and high concentrations of omega-agatoxin-IVA (Q-type). The P-type current, sensitive to low concentrations of omega-agatoxin-IVA, transiently increased between E15 and E17, and then both current density and its proportion of the global current decreased. 4. Our results reveal large modifications in the expression of voltage-dependent calcium channels during embryonic development of primary vestibular neurons. The changes in the expression of LVA current and the transient augmentation of P-type HVA current occur during a period characterized by massive neuronal growth and by the beginning of synaptogenesis. These results suggest a specific role of these currents in the ontogenesis of vestibular primary afferents.

Calcium Currents of Rhythmic Neurons Recorded in the Isolated Respiratory Network of Neonatal Mice

To obtain a quantitative characterization of voltage-activated calcium currents in respiratory neurons, we performed voltage-clamp recordings in the transverse brainstem slice of mice from neurons located within the ventral respiratory group. It is assumed that this medullary region contains the neuronal network responsible for generating the respiratory rhythm. This study represents one of the first attempts to analyze quantitatively the currents in respiratory neurons. The inward calcium currents of VRG neurons consisted of two components: a high voltage-activated (HVA) and a low voltage-activated (LVA) calcium current. The activation threshold of the HVA current was at -40 mV. It was fully activated (peak voltage) between -10 and 0 mV. The half-maximal activation (V50) was at -27. 29 mV +/- 1.15 (n = 24). The HVA current was inactivated completely at a holding potential of -35 mV and fully deinactivated at a holding potential of -65 mV (V50, -52.26 mV +/- 0.27; n = 18). The threshold for the activation of the LVA current was at -65 mV. This current had its peak voltage between -50 and -40 mV (mean, V50 = -59. 15 mV +/- 0.21; n = 15). The LVA current was inactivated completely at a holding potential of -65 mV and deinactivated fully at a holding potential of -95 mV (mean, V50 = -82.40 mV +/- 0.32; n = 38). These properties are consistent with other studies suggesting that the LVA current is a T-type current. The properties of these inward currents are discussed with respect to their role in generating Ca2+ potentials that may contribute to the generation of the mammalian respiratory rhythm.

Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones

1. Whole-cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). 2. Both high voltage-activated (HVA) and low voltage-activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. 3. The transient LVA calcium current activated rapidly on depolarization (half-activation, -59 mV) and inactivated during maintained depolarization (half-inactivation, -89 mV). This T-type current was observed in somatic recordings but was absent from presynaptic terminals. 4. On the basis of their pharmacological sensitivity, P/Q-type Ca2+ channels accounted for only 6 % of the somatic HVA, while L-, N- and R-type Ca2+ channels each accounted for around one-third of the somatic calcium current. 5. The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. 6. We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P-type contribute little to somatic currents but predominate at the synaptic terminal; L-, N- and R-types dominate at the soma, but contribute negligibly to calcium currents in the terminal.

Differential regulation of potassium currents by FGF-1 and FGF-2 in embryonic Xenopus laevis myocytes.

1. Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage-dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF-1 and FGF-2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos. 2. Long-term exposure of the embryonic myocytes to FGF-1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage-dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF-2 upregulated these currents, although, because of an increase in capacitance caused by FGF-2, current density did not change with this factor. 3. The regulation of IK(IR) by FGF-1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF-1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel-blocking mechanism. 4. In many cells, IK, S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage-activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S. 5. In summary, FGF-1 and FGF-2 exert opposite effects on voltage-dependent K+ currents in embryonic myocytes and, furthermore, FGF-1 achieves its effects on different K+ currents via separate second messenger pathways.

The effect of overexpression of auxiliary Ca2+ channel subunits on native Ca2+ channel currents in undifferentiated mammalian NG108-15 cells.

1. High voltage activated (HVA) Ca2+ channels are composed of a pore-forming alpha 1 subunit and the accessory beta and alpha2-delta subunits. However, the subunit composition of low voltage activated (LVA), or T-type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2. Endogenous alpha 1A, B, C, C, and E, and low levels of beta and alpha 2-delta subunit protein were demonstrated in undifferentiated NG108-15 cells. 3. The alpha 2-delta, beta 2a or beta 1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of alpha 2-delta but also of beta 2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above -30 mV, and alpha 2-delta additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T-type currents. In contrast, beta 1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108-15 cells, overexpression of the Ca2+ channel accessory subunits alpha 2-delta and beta 2a induce a sustained component of HVA current, and alpha 2-delta also influences the voltage dependence of activation of the LVA current. It is possible that native T-type alpha 1 subunits are not associated with beta subunits.

Conopressin affects excitability, firing, and action potential shape through stimulation of transient and persistent inward currents in mulluscan neurons.

The molluscan vasopressin/oxytocin-related neuropeptide conopressin activates two persistent inward currents in neurons from the anterior lobe of the right cerebral ganglion of Lymnaea stagnalis that are involved in the control of male copulatory behavior. The low-voltage-activated (LVA) current is activated at a wide range of membrane potentials, its amplitude being only weakly voltage dependent. The high-voltage-activated (HVA) current is activated at potentials positive to -40 mV only and shows a steep voltage dependence. Occurrence of both currents varies from cell to cell, some expressing both and others only the HVA current. In most neurons that have the LVA current, a conopressin-independent persistent inward current (INSR) is found that resembles the HVA current in its voltage dependence. The functional importance of the LVA and HVA currents was studied under current-clamp conditions in isolated anterior lobe neurons. In cells exhibiting both current types, the effect of activation of the LVA current alone was investigated as follows: previously recorded LVA current profiles were injected into the neurons, and the effects were compared with responses induced by conopressin. Both treatments resulted in a strong depolarization and firing activity. No differences in firing frequency and burst duration were observed, indicating that activation of the LVA current is sufficient to evoke bursts. In cells exhibiting only the HVA current, the effect of conopressin on the response to a depolarizing stimulus was tested. Conopressin reversibly increased the number of action potentials generated by the stimulus, suggesting that the HVA current enhances excitability and counteracts accommodation. Conopressin enhanced action potential broadening during depolarizing stimuli in many neurons. Voltage-clamp experiments performed under ion-selective conditions revealed the presence of transient sodium and calcium currents. Using the action potential clamp technique, it was shown that both currents contribute to the action potential. The calcium current, which is activated mainly during the repolarizing phase of the action potential, is augmented by conopressin. Thus conopressin may directly modulate the shape of the action potential. In summary, conopressin may act simultaneously on multiple inward currents in anterior lobe neurons of Lymnaea to affect firing activity, excitability, and action potential shape.

Voltage-Dependent Calcium Currents in Bulbospinal Neurons of Neonatal Rat Rostral Ventrolateral Medulla: Modulation by α2-Adrenergic Receptors

The properties and modulation by norepinephrine (NE) of voltage-dependent calcium currents were studied in bulbospinal neurons (n = 116) of the rostral ventrolateral medulla (RVLM) using whole cell patch-clamp techniques in neonatal rat brain stem slices. RVLM bulbospinal neurons were identified visually by their location in slices and by the presence of flourescein isothiocyanate-tagged microbeads, which were injected into the spinal cord before the experiment; RVLM neurons were filled with Lucifer yellow during recordings, and the slice was processed for detection of tyrosine hydroxylase immunoreactivity (TH-IR). Thirty-four of 42 recovered cells (81%) were positive for TH-IR, indicating that most recorded cells were C1 neurons. Bulbospinal RVLM neurons expressed a prominent high-voltage-activated (HVA) calcium current, which began to activate at -30 to -40 mV (from a holding potential of -60 or -70 mV), and peaked at approximately 0 mV (0.8 +/- 0.1 nA;mean +/- SE). HVA current comprised predominantly omega-conotoxin GVIA-sensitive, N-type and omega-agatoxin IVA-sensitive, P/Q-type components, with smaller dihydropyridine-sensitive, L-type, and residual current components. Most RVLM bulbospinal neurons (n = 44/52, including 12/14 histologically identified C1 cells) also expressed low-voltage-activated (LVA) calcium current. LVA current began to activate at approximately -60 mV (from a holding potential of -100 mV) and was nearly completely inactivated at -50 mV with a half-inactivation potential of -70 +/- 2 mV. The amplitude of LVA current at -50 mV was 78 +/- 24 pA with Ba2+ and 156 +/- 38 pA with Ca2+ as a charge carrier. NE inhibited HVA current in most bulbospinal RVLM neurons (n = 70/77) with an EC50 of 1.2 muM; NE had no effect on LVA current. Calcium current inhibition by NE was mediated by alpha2-adrenergic receptors (alpha2-ARs) as the effect was mimicked by the selective alpha2-AR agonist, UK-14,304, and blocked by idazoxan, an alpha2-AR antagonist, but unaffected by prazosin and propranolol (alpha1- and beta-AR antagonists, respectively). Most of the NE-sensitive calcium current was N- and P/Q-type. NE-induced inhibition of calcium current evoked by action potential waveforms (APWs) was significantly larger than that evoked by depolarizing steps (34 +/- 2.5 vs. 23 +/- 2.7%; P < 0.05). Although inhibition of calcium current was voltage dependent and partially relieved by strong depolarizations, when calcium currents were evoked with a 10-Hz train of APWs as a voltage command, the inhibitory effect of NE was maintained throughout the train. In conclusion, bulbospinal RVLM neurons, including C1 cells, express multiple types of calcium currents. Inhibition of HVA calcium current by NE may modulate input-output relationships and release of transmitters from C1 cells.