Low Voltage-activated Channels Research Articles

Roles of Molecular Regions in Determining Differences between Voltage Dependence of Activation of CaV3.1 and CaV1.2 Calcium Channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.

Pharmacology of recombinant low-voltage activated calcium channels.

Several types of voltage- or ligand-activated calcium channels contribute to the excitability of neuronal cells. Low-voltage-activated (LVA), T-type calcium channels are characterised by relatively negative threshold of activation and therefore they can generate low-threshold spikes, which are essential for burst firing. At least three different proteins form T-type calcium current in neurons: Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. Expression of these proteins in various brain regions is complementary. Individual channel types could be distinguished by different sensitivity towards inorganic cations. This inhibition can contribute to the toxicity of some heavy metals. Selective inhibition of T-type calcium channels by organic blockers may have clinical importance in some forms of epilepsy. Mibefradil inhibits the expressed Ca(v2)3.1, Ca(v)3.2 and Ca(v)3.3 channels in nanomolar concentrations with Ca(v)3.3 channel having lowest affinity. The sensitivity of the expressed Ca(v)3.1 channel to the antiepileptic drugs, valproate and ethosuximide, is low. Ca(v)3.1 channel is moderately sensitive to phenytoin. The Ca(v)3.2 channel is sensitive to ethosuximide, amlodipine and amiloride. All three LVA calcium channels are moderately sensitive to active metabolites of methosuximide, i.e. alpha-methyl-alpha-phenylsuccinimide. Several neuroleptics inhibit all three LVA channels in clinically relevant concentrations. All three channels are also inhibited by the endogenous cannabinoid anandamide. A high affinity peptide blocker for these Ca channels is the scorpion toxin kurtoxin which inhibits the Ca(v)3.1 and Ca(v)3.2, but not the Ca(v)3.3 channel in nanomolar concentrations. Nitrous oxide selectively inhibits the Ca(v)3.2, but not the Ca(v)3.1 channel. The Ca(v)3.2, but not the Ca(v)3.1 channel is potentiated by stimulation of Ca(2+)/CaM-dependent protein kinase.

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca(2+)-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca(2+) channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell's periphery. HT1080 cells exhibit periodic intracellular Ca(2+) spikes. High-speed imaging revealed that the Ca(2+) spikes were composed of a single Ca(2+) wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca(2+) channel blocker mibefradil inhibited Ca(2+) spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd(3+) and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca(2+) channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca(2+) wave, thereby enhancing the efficacy of emerging therapeutic protocols.

Preservation of motor neuron Ca2+ channel sensitivity to insulin-like growth factor-1 in brain motor cortex from senescent rat.

Despite the multiple effects on mammals during development, the effectiveness of the insulin-like growth factor-1 (IGF-1) to sustain cell function and structure in the brain of senescent mammals is almost completely unknown. To address this issue, we investigated whether the effects of IGF-1 on specific targets are preserved at later stages of life. Voltage-gated Ca2+ channels (VGCC) are well-characterized targets of IGF-1. VGCC regulate membrane excitability and gene transcription along with other functions that have been found to be impaired in the brain of senescent rodents. As the voluntary control of movement has been reported to be altered in the elderly, we investigated the expression, function and responsiveness of high (HVA)- and low-voltage-activated (LVA) Ca2+ channels to IGF-1, using the whole-cell configuration of the patch-clamp and RT-PCR in the specific region of the rat motor cortex that controls hindlimb muscle movement. We detected the expression of alpha 1A, alpha 1B and alpha 1E genes encoding the HVA Ca2+ channels P/Q, N and R, respectively, but not alpha 1C, alpha 1D, alpha 1S encoding the L-type Ca2+ channel in this region of the brain cortex. IGF-1 enhanced Ca2+ channel currents through P/Q- and N-type channels but not significantly through the R-type or LVA channels. IGF-1 enhanced the amplitude but did not modify the voltage dependence of Ca2+ channel currents in young (2- to 4-week-old), young adult (7-month-old) and senescent (28- to 29-month-old) rats. These results support the concept that despite the reported decrease in circulating (liver) and local (central nervous system) production of IGF-1 with ageing, key neuronal targets such as the VGCC remain responsive to the growth factor throughout life.

T-type calcium channel regulation by specific G-protein betagamma subunits.

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

Molecular physiology of low-voltage-activated t-type calcium channels.

T-type Ca2+ channels were originally called low-voltage-activated (LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons Ca2+ influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials mediated by Na+ channels. Burst firing is thought to play an important role in the synchronized activity of the thalamus observed in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In addition to a pacemaker role, Ca2+ entry via T-type channels can directly regulate intracellular Ca2+ concentrations, which is an important second messenger for a variety of cellular processes. Molecular cloning revealed the existence of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found in high-voltage-activated (HVA) Ca2+ channels, and Na+ channels, indicating that they are evolutionarily related. Hence, the alpha1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The electrophysiological activities of recombinant Cav3 channels are very similar to native T-type currents and can be differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and smaller conductance of Ba2+. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by Ni2+. The goal of this review is to provide a comprehensive description of T-type currents, their distribution, regulation, pharmacology, and cloning.

Functional Interaction between Mouse Spermatogenic LVA and Thapsigargin-Modulated Calcium Channels

The acrosome reaction in mouse is triggered by a long-lasting calcium signaling produced by a chain of openings of several calcium channels, a low-voltage-activated (LVA) calcium channel, an inositol trisphosphate receptor (IP 3R), and the store-operated calcium channel TRP2. Since mature sperm cells are refractory to patch clamp experiments, we study the functional interactions among those sperm calcium channels in spermatogenic cells. We have studied the role of cytosolic calcium in voltage-dependent facilitation of low voltage-activated calcium channels. Calcium concentration was modified through the inclusion of the calcium buffers, EGTA and BAPTA, in the recording pipette solution, and by addition of calcium modulators like thapsigargin and the calcium ionophore A23187. We demonstrate that lowering calcium concentration below resting level allows to evidence a voltage-dependent facilitation. We also show that LVA calcium channels present strong voltage-dependent inhibition by thapsigargin. This effect is independent of cytosolic calcium elevation secondary to calcium store depletion and to the activation of TRP channels. Our data evidence an interesting functional relationship, in this cell type, between LVA channels and proteins whose activity is related to calcium filling state of the endoplasmic reticulum (presumably TRP channels and inositol triphosphate receptor). These relationships may contribute to the regulation of calcium signaling during acrosome reaction of mature sperm cell.

Cloning and Expression of the Human T-Type Channel Cav3.3: Insights into Prepulse Facilitation

The full-length human Cav3.3 (α1I) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3′ untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5mM Ca2+ as charge carrier. From a holding potential of −100mV, step depolarizations elicited inward currents with an apparent threshold of −70mV, a peak of −30mV, and reversed at +40mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Cav3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Cav3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Cav3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100mV, 200ms) increased the fraction of channels that gated at low voltages. In contrast, human Cav3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Cav3.3 is similar to high voltage-activated Ca2+ channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.

Cardiac function involving the T-type Ca2+ channel

Functional involvement of the T-type calcium channel in the heart excitation at the pathophysiological conditions has been elucidated. The T-type channel is classified as the low voltage-activated channel (LVA) solely in the voltage activated calcium channel. In 1998, Perez-Reyes and coworkers cloned the first LVA alpha(1) subunit, which was named as alpha(1G) or Ca(V)3.1. In the cardiac muscle, alpha(1G) and the other clone alpha(1H) are dominantly expressed in the sinoatrial node, atrioventricular node and other signal conduction tissues. Abnormal activity of T channels has been suggested in the following cardiovascular diseases: hypertension, cardiac hypertrophy, cardiac infarction. The cloning of the T-type calcium channel allows us to understand the function of the channel in detail and options for therapeutics in the T-type channel-related diseases.

Vascular Smooth Muscle Calcium Channels

Voltage-gated Ca2+ channels of the plasma membrane open in response to changes in cell membrane potential, providing an important pathway for regulated entry of extracellular Ca2+ ions. Ca2+ channels comprise a family of structurally related proteins displaying tissue- and stage-specific expression. They are classified as either high voltage-activated (HVA) or low voltage-activated (LVA) and can be subdivided further based on their pharmacological properties. HVA channels include L-, N-, P-/Q-, and R-types, and LVA channels are designated as T-type. Members of each subtype have been identified by molecular cloning, facilitating studies of their biophysical properties, relative distribution, and contribution to Ca2+-dependent cellular functions. In recent years, it has become increasingly clear that in most, if not all, cases, more than one class of voltage-dependent Ca2+ channel are expressed in a given cell type. The functional relevance of different Ca2+ channels within a cell, however, remains unclear. In contractile cells, voltage-gated Ca2+ channels are particularly important because influx of extracellular Ca2+ is essential for muscle contraction and maintenance of tension. Ca2+ channels have received a great deal of attention in vascular smooth muscle (VSM), where calcium influx affects arterial vasoconstriction and vasorelaxation, and ultimately influences systemic blood pressure. Resistance arteries are an important site of blood pressure regulation. These arteries normally exist in a relatively contracted state and can undergo either dilation or constriction, depending on the contractile state of the arterial VSM. Ca2+ channel activity is a key determinant of VSM contractile state, and L-type Ca2+ channels are the classic target of calcium channel blockers (CCBs) widely prescribed as antihypertensive drugs. Therefore, a wealth of information has been published comparing different calcium antagonists, or CCBs, and their inhibitory affects on L-type calcium currents and vasoconstriction. It is important to …

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.

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II.

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).

All classes of calcium channel couple with equal efficiency to exocytosis in rat melanotropes, inducing linear stimulus-secretion coupling.

1. The contribution of low voltage-activated (LVA) T-type Ca2+ channels and four different types of high voltage-activated (HVA) Ca2+ channel to exocytosis, and the relationship between calcium influx and exocytosis during action potentials (APs) were studied in pituitary melanotropes. 2. Selective HVA Ca2+ channel blockers reduced exocytosis, monitored by membrane capacitance measurements, proportional to the reduction in Ca2+ influx. The efficacy of Ca2+ in stimulating exocytosis did not change in the presence of the Ca2+ channel blockers, indicating that all HVA Ca2+ channels act together in stimulating exocytosis. 3. The relationship between Ca2+ influx and exocytosis during the AP was examined using APs recorded from spontaneously active melanotropes as command templates under voltage clamp. Under voltage clamp, multiphasic Ca2+ currents were activated over the entire duration of the APs, i.e. during the rising phase as well as the plateau phase. The maximum amplitude of the Ca2+ current coincided with the peak of the AP. 4. The relationship between Ca2+ entry and exocytosis was linear for the different phases of the AP. Also, the influx of Ca2+ through LVA T-type channels stimulated exocytosis with the same efficacy as through the HVA channels. 5. APs of increasing duration ( approximately 50 to approximately 300 ms) evoked increasing amounts of exocytosis. The number of entering Ca2+ ions and the capacitance change were linearly related to AP duration, resulting in a fixed relationship between Ca2+ entry and exocytosis. 6. The results show that Ca2+ ions, entering a melanotrope, couple with equal strength to exocytosis regardless of the channel type involved. We suggest that the linear relationship between Ca2+ entry and secretion observed under physiological conditions (during APs), results from the equal strength with which LVA and HVA channels in melanotropes couple to exocytosis. This guarantees that secretion takes place over the entire duration of the AP.

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.

Cloning and Functional Expression of a Voltage-gated Calcium Channel α1 Subunit from Jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.

Antisense oligonucleotides against rat brain alpha1E DNA and its atrial homologue decrease T-type calcium current in atrial myocytes.

Low voltage-activated, or T-type, calcium currents are important regulators of neuronal and muscle excitability, secretion, and possibly cell growth and differentiation. The gene (or genes) coding for the pore-forming subunit of low voltage-activated channel proteins has not been unequivocally identified. We have used reverse transcription-PCR to identify partial clones from rat atrial myocytes that share high homology with a member of the E class of calcium channel genes. Antisense oligonucleotides targeting one of these partial clones (raE1) specifically block the increase in T-current density that normally results when atrial myocytes are treated with insulin-like growth factor 1 (IGF-1). Antisense oligonucleotides targeting portions of the neuronal rat alpha1E sequence, which are not part of the clones detected in atrial tissue, also block the IGF-1-induced increase in T-current, suggesting that the high homology to alpha1E seen in the partial clone may be present in the complete atrial sequence. The basal T-current expressed in these cells is also blocked by antisense oligonucleotides, which is consistent with the notion that IGF-1 up-regulates the same gene that encodes the basal current. These results support the hypothesis that a member of the E class of calcium channel genes encodes a low voltage-activated calcium channel in atrial myocytes.

Characterization of single voltage‐gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons.

1. We have used dendrite-attached patch-clamp techniques to record single Na+ and Ca2+ channel activity from the apical dendrites (up to 350 microns away from soma) of CA1 pyramidal neurons in rat hippocampal slices (ages: 2-8 weeks). 2. Na+ channels were found in every patch examined (range: 2 to > 20 channels per patch). Channel openings, which had a slope conductance of 15 +/- 0.3 pS (mean +/- S.E.M.), began with test commands to around -50 mV and consisted of both early transient channel activity and also later occurring prolonged openings of 5-15 ms. All Na+ channel activity was suppressed by inclusion of TTX (1 microM) in the recording pipette. 3. Ca2+ channel activity was recorded in about 80% of the patches examined (range: 1 to > 10 channels per patch). Several types of channel behaviour were observed in these patches. Single channel recordings in 110 mM BaCl2, revealed an approximately 10 pS channel of small unitary current amplitude (-0.5 pA at -20 mV). These channels began activating at relatively hyperpolarized potentials (-50 mV) and ensemble averages of this low voltage-activated (LVA) channel activity showed rapid inactivation. 4. A somewhat heterogeneous population of high voltage-activated, moderate conductance (HVAm; approximately 17 pS), Ca2+ channel activity was also encountered. These channels exhibited a relatively large unitary amplitude (-0.8 pA at 0 mV) and ensemble averages demonstrated moderate inactivation. The HVAm population of channels could be tentatively subdivided into two separate groups based upon mean channel open times. 5. Less frequently, HVA, large conductance (27 pS) Ca2+ channel activity (HVA1) was also observed. This large unitary amplitude (-1.5 pA at 0 mV) channel activity began with steps to approximately 0 mV and ensemble averages did not show any time-dependent inactivation. The dihydropyridine Ca2+ channel agonist Bay K 8644 (0.5 or 1 microM) was found to characteristically prolong these channel openings. 6. omega-Conotoxin MVIIC (10 microM), did not significantly reduce the amount of channel activity recorded from the LVA, HVAm or HVA1 channel types in dendritic patches. In patches from somata, omega-conotoxin MVIIC was effective in eliminating a significant amount of HVAm Ca2+ channel activity. Inclusion of 50 or 100 microM NiCl2 to the recording solution significantly reduced the amount of channel activity recorded from LVA and HVAm channel types in dendritic patches. A subpopulation of HVAm channels was, however, found to be Ni2+ insensitive. Dendritic HVA, channel activity was unaffected by these low concentrations of Ni2+.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

T-type Ca2+ channels are required for adrenocorticotropin-stimulated cortisol production by bovine adrenal zona fasciculata cells.

The function of low voltage-activated T-type Ca2+ channels in ACTH-stimulated cortisol production by bovine adrenal zona fasciculata cells (AZF) was explored in patch clamp and secretion studies. Nearly all AZF cells expressed only a low voltage-activated T-type Ca2+ current (IT) that was blocked by the diphenylbutylpiperidine (DPBP) Ca2+ antagonists penfluridol and pimozide with IC50S of 0.3 and 0.5 microM, respectively. Dihydropyridine (DHP) Ca2+ antagonists, including nimodipine, nisoldipine, and felodipine, also blocked T-type Ca2+ current with IC50S ranging from 3.5-8.8 microM. Inhibition of IT by DPBP and DHP antagonists was voltage and use dependent. ACTH (1 nM) stimulated large (> 50-fold) increases in cortisol production by AZF cells, which were inhibited by Ca2+ antagonists at concentrations similar to those which blocked IT. Inhibition of cortisol production by Ca2+ antagonists was specific; ACTH-induced insulin-like growth factor-I production by AZF cells was not affected by DPBP antagonists. The L channel-specific DHP Ca2+ agonist (-)Bay K 8644 did not enhance basal or ACTH-stimulated cortisol synthesis. These results demonstrate that functional T-rather than L-type Ca2+ channels are required for ACTH-stimulated cortisol synthesis. They also suggest that these low voltage-activated channels, acting as the primary pathway for Ca2+ entry into AZF cells, couple ACTH-stimulated membrane depolarization to steroid hormone production.

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.