Low Voltage-activated Channels Research Articles

Interaction of MDIMP with the Voltage-Gated Calcium Channels.

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC50 values were found (in micromolars): >1000 (CaV2.3), 957 (CaV1.3), 656 (CaV1.2), 219 (CaV3.2), and 132 (CaV3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca2+ channels and a modest upregulation of CaV1.3 and CaV2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning CaV3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC50 value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (R)-MDIMP [methyl (R)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca2+ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: coexistence of a non-selective cationic channel and of T-type Ca channels.

In rat pulmonary vein (PV) cardiomyocytes (CM), norepinephrine (NE) induces an automatic activity consisting of bursts of slow action potentials which depend on Ca2+ (upstroke) and Na+ (inter-burst) channels. Our objective was to characterize low voltage-activated (LVA) currents in rat PVCM susceptible to trigger this activity. Whole-cell ICa (5 mM Ca2+) was recorded from - 100 mV with classical Na+- and K+-free solutions. A fast LVA ICa (FLVA-ICa), present in ≈ 56% of PVCM between ~ - 50 to - 20 mV, was blocked by 10 μM TTX and markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P'Ca/PNa and P'Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 and 1.88 ± 0.25, and not different from a value of 2. FLVA-ICa was increased by 10 μM NE and 300 nM BayK8644, decreased by 5 μM nifedipine but not blocked by ranolazine (10 μM). NiCl2 (40 μM) and TTA-A2 (10 or 100 nM) increased FLVA-ICa. Similar results were obtained in left atrial (LA) CM. Neither Ba2+ nor Sr2+ alone could permeate the FLVA channel or block Ca2+ influx but revealed a large slower activating and inactivating LVA Ca2+ current (SLVA-ICa), present in 10 out of 80 PVCM, absent in LACM, and partially inhibited by 100 nM TTA-A2. Therefore, the ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel with a dihydropyridine binding site. SLVA-ICa might correspond to Ca2+ influx through Cav3.x channels and contribute to triggering NE-induced automatic activity in the PV myocardial sleeve.

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion.

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells.

Low voltage‐activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse

A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca(2+) entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (-50 mV) or low-concentration Ni(2+) (0.1 mM). Transmission persisted using a voltage pulse to -40 mV from a holding potential of -80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni(2+)-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.

Mice mechanoreceptors are modulated by Cav3.2 T‐type calcium channels

Mechanoreceptors are sensory receptors that respond to mechanical displacement and provide the senses of touch, pressure, vibration and proprioception. Mice have three types of low-threshold mechanoreceptors: slowly adapting mechanoreceptors (SAMs), rapidly adapting mechanoreceptors (RAMs) and D-hair receptors (Driscoll & Tavernarakis, 2000). D-hair receptors are the end-terminal of type Aδ fibre axons and present a high sensitivity and ultra-rapid temporal detection of slowly moving stimuli. The mRNA expression for the T-type voltage-gated calcium channel Cav3.2 is the only marker defined for mechanoreceptors and found almost exclusively in D-hair receptors. The Cav3.2 gene encodes one of the three low-voltage-activated (LVA) Ca2+ channels (Cav3.1, Cav3.2 and Cav3.3) and are all expressed by sensory neurons in the DRG (Iftinca et al. 2007). Previous published studies proposed that Cav3.2 channel has a primary role in nociception (Bourinet et al. 2005). However, another research group that used miberfradil, a drug that blocks all T-type channels, suggested that the mechanosensitivity of D-hair receptors is reduced by blockade of LVA channels (Shin et al. 2003). For this reason the specific physiological properties of the Cav3.2 channel has not been accurately described. In a recent issue of The Journal of Physiology, Wang & Lewin (2011) conducted a study with the purpose of demonstrating the role of the T-type Cav3.2 channels. In this study, Cav3.2−/− mutant mice were used to directly address the role of this channel in sensory transduction of cutaneous mechanoreceptors and nociceptors. Mice were produced by gene targeting in mouse ES cells; the targeting vector was designed to delete the IS5 region of the murine Cacna1H gene, which resulted in deletion of exon 6, corresponding to amino acid residues 216 to 267. This method was helpful in examining the mechanosensitivity of nociceptors and mechanoreceptors in normal Cav3.2 mice and in Cav3.2−/− mutant mice, proving that nociceptors sensitivity was not altered when the Cav3.2 gene was suppressed; on the other hand, D-hair receptor sensibility was drastically impaired in their ability to encode the properties of moving stimuli, losing their high sensitivity and ability to ensure rapid temporal detection to skin movement. In addition the functional properties of other mechanoreceptors and nociceptors were unaffected by the suppression of Cav3.2 channels. Why is this important? These new data provide conclusive results of the specific function of the T-type Cav3.2 channel, solving the previous controversy about its physiological function and proving that this specific gene is responsible for providing the functional and distinctive properties of D-hair receptors. Thus, this study encloses the specific activity of this gene, being now considered not only a high specific marker of D-hair receptors but also indispensable to maintain their high sensitivity in the detection of skin deflection. The expression of Cav3.2 in D-hair mechanoreceptors may serve to provide highly accurate temporal information about rapidly changing stimuli. This information helps humans to make fast and precise judgments about the speed and direction of tactile stimuli, a characteristic that can be considered vital for survival and functionality. This is the first time that the regulation for temporal coding in mouse mechanoreceptors from Cav3.2 T-type channels has been proved. Nevertheless, results from this study showed that there exists a possibility that a minor sub-population of nociceptors is dependent on the presence of Cav3.2 channels. The research revealed no certainty that such a population exists, and it is highly unlikely that this kind of population could be identified. Further studies will be needed to ascertain if this is indeed a possibility.

Alteration in voltage-dependent calcium channels in dog basilar artery after subarachnoid hemorrhage

The L-type Ca++ channel antagonists like nimodipine have limited efficacy against vasospasm after subarachnoid hemorrhage (SAH). The authors tested the hypothesis that this is because SAH alters these channels, rendering them less responsible for contraction. Basilar artery smooth muscle cells were isolated 4, 7, and 21 days after SAH in dogs, and Ca++ channel currents were recorded in 10-mmol/L barium. Proteins for α₁ subunits of L-type Ca++ channels were measured by immunoblotting and isometric tension recordings done on rings of the basilar artery. High voltage-activated (HVA) Ca++ channel currents were significantly decreased and low voltage-activated (LVA) currents increased during vasospasm 4, 7, and 21 days after SAH (p < 0.05). Vasospasm was associated with a significant decrease in the number of cells with negligible LVA current while the number of cells in which the LVA current formed greater than 50% of the maximal current increased (p < 0.01). Window currents through LVA and HVA channels were significantly reduced. All changes correlated with the severity of vasospasm. There was an increase in protein for Ca(v)3.1 and Ca(v)3.3 α₁ subunits that comprise T-type Ca++ channels, a decrease in L-type (Ca(v)1.2 and Ca(v)1.3) and an increase in R-type (Ca(v)2.3) Ca++ channel α₁ subunits. Functionally, however, isometric tension studies showed vasospastic arteries still relaxed with nimodipine. Voltage-dependent Ca++ channels are altered in cerebral arteries after SAH. While decreased L-type channels may account for the lack of efficacy of nimodipine clinically, there may be other reasons such as inadequate dose, effect of nimodipine on other cellular targets, and mechanisms of vasospasm other than smooth muscle contraction mediated by activation of L-type Ca++ channels.

Regulation of Ca v 3.1 channels by glucocorticoids.

The activity of low voltage-activated Ca(2+) (Ca(V)3) channels is tightly coupled to neurotransmitter and hormone secretion. Previous studies have shown that Ca(V)3 channels are regulated by glucocorticoids (GCs), though the mechanism underlying channel regulation remains unclear. Here, using the pituitary GH(3) cell line as a model, we investigated whether Ca(V)3 channel expression is under the control of GCs, and if their actions are mediated by transcriptional and/or post-transcriptional mechanisms. RT-PCR and western blot analyses showed that Ca(V)3.1 but not Ca(V)3.2 and Ca(V)3.3 channels is expressed in the GH(3) cells, and patch clamp recordings confirmed that Ca(2+) currents through low voltage-activated channels were decreased after chronic treatment with GCs. Consistent with this, total plasma membrane expression of Ca(V)3.1 protein as analyzed by cell-surface biotinylation assays and semi-quantitative western blotting was also down-regulated, while quantitative real-time RT-PCR analysis revealed a significant decrease of Ca(V)3.1 mRNA expression in the treated cells. In contrast, patch-clamp recordings on HEK-293 cells stably expressing recombinant Ca(V)3.1 channels showed that Ca(2+) currents were not affected by GC treatment. These results suggest that decreased transcription is a likely mechanism to explain the inhibitory actions of GCs on the functional expression of native Ca(V)3.1 channels.

Protein Kinase A Activity Controls the Regulation of T-type CaV3.2 Channels by Gβγ Dimers

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gbetagamma dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gbeta(2)gammax dimers to effect voltage-independent inhibition of Ca(v)3.2 channels. Inhibition requires phosphorylation of Ser(1107), a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gbeta(2)gamma(2) dimers but does not disrupt dimer binding nor change its specificity. Gbetagamma dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Ca(v)3.2 whole cell current is precluded by Gbetagamma-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D(1) or D(2) agonists do not elicit channel inhibition yet together synergize to inhibit Ca(v)3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Ca(v)3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

A Small Peptide Inhibitor of the Low Voltage-Activated Calcium Channel Cav3.1

The calcium channel gamma(6) subunit modulates low voltage-activated (LVA) calcium current in both human embryonic kidney (HEK) cells and cardiomyocytes, although the mechanism of modulation is unknown. We recently showed that gamma(6) contains a critical GxxxA motif in the first transmembrane domain (TM1) that is essential for its inhibition of the Cav3.1 (LVA) calcium current. In this study, we tested the hypothesis that an eight-amino acid peptide that contains the GxxxA motif from gamma(6) TM1 can act as a novel pharmacological inhibitor of the Cav3.1 calcium current by performing whole-cell electrophysiology. Our results demonstrate that the peptide inhibits Cav3.1 current by dynamically binding and dissociating from the Cav3.1 channel in a concentration-dependent but largely voltage-independent manner. By selectively substituting residues within the peptide, we show that both the GxxxA framework and surrounding aliphatic side-chains contribute to the presumably interhelical interactions between gamma(6) TM1 and the Cav3.1 channel. The fast kinetics of the interaction supports the view that gamma(6) acts as an endogenous LVA channel antagonist within the plasma membrane, suggesting a mechanism other than regulation of surface expression or membrane trafficking of the pore-forming subunit of the channel. We also demonstrate that the peptide has different affinities for Cav3.1 and Cav1.2 calcium currents, which is consistent with the selective effect of gamma(6) on LVA and high voltage-activated calcium currents in vivo.

Characterization of the Gating Brake in the I-II Loop of Cav3.2 T-type Ca2+ Channels

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

The Ca V3.1 T-type Ca 2+channel contributes to voltage-dependent calcium currents in rat outer hair cells

Calcium currents through voltage-dependent Ca 2+channels (VDCCs) in mammalian outer hair cells (OHCs) are generally considered to possess the pharmacological properties of L-type (dihydropyridine-sensitive) currents. However, the OHCs' low resting potentials and their slight depolarization upon sound stimuli suggest that the low voltage-activated channels may contribute to Ca 2+regulation. We present morphological and electrophysiological evidence for the presence of the Ca V3.1 T-type Ca 2+channels, one of the low voltage-activated Ca 2+channels, in mature rat OHCs. PCR experiments revealed the expression of Ca V3.1, but not Ca V3.2 or Ca V3.3, in the mature rat cochlea. In situ hybridization and immunohistochemistry revealed expression of Ca V3.1 in both inner and outer hair cells at the mRNA level, but only in the OHCs at the protein level. Western blot analysis of anti-Ca V3.1 antibody showed a 242 kDa band in mature rat cochlear lysates. Patch-clamp recordings of OHCs isolated from rat cochleae after the onset of hearing revealed that whole-cell voltage-dependent Ca 2+ currents were significantly increased in depolarizing steps from a holding potential of − 100 mV when compared with those from − 70 mV. Only the currents from − 100 mV manifested a distinct transient inward Ca 2+ current, and this transient component was effectively blocked by 1 μM of the T-type-specific antagonist, mibefradil. Our data suggest an involvement of Ca V3.1 in intracellular Ca 2+regulation in mature OHCs.

Molecular Determinant for Specific Ca/Ba Selectivity Profiles of Low and High Threshold Ca2+ Channels

Voltage-gated Ca2+ channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca2+ over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca2+ ions. This locus however does not explain either the choice of Ca2+ among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca2+ and Ba2+ reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The CaV2.1 selectivity profile is transferred to CaV2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca2+ binding site in the channel pore directly involved in the choice of Ca2+, among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca2+ influx between the different HVA channels.

Voltage-Dependent Calcium Currents Are Enhanced in Nucleus of the Solitary Tract Neurons Isolated From Renal Wrap Hypertensive Rats

The nucleus of the solitary tract (NTS) is the central site of termination of baroreceptor afferents. We hypothesize that changes occur in voltage-gated calcium channels (VGCCs) within NTS neurons as a consequence of hypertension. Whole-cell patch-clamp recordings were obtained from adult normotensive (109+/-2 mm Hg; n=6 from 6 sham-operated and 31 nonsurgically treated) and hypertensive (158+/-6 mm Hg; n=24) rats. In some experiments, 4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide was applied to the aortic nerve to visualize NTS neurons receiving baroreceptor synaptic contacts. Ba(2+) currents (500 ms; -80 mV prepotential; 500 ms voltage steps in 5-mV increments to +15mV) peaked between -20 and -10 mV and were blocked by 100 mum of Cd(2+). Peak VGCCs were not different comparing non-4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled and 4-(4- [dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled NTS neurons in hypertensive and normotensive rats. The peak VGCC was significantly greater in cells from hypertensive compared with normotensive rats for both non-DiA-labeled (P=0.02) and DiA-labeled (P=0.04) neurons. To separate high-voltage activated (HVA) and low-voltage activated (LVA) components of VGCCs, voltage ramps (-110 mV to +30 mV over 50 ms) were applied from a holding potential of -60 mV (LVA channels inactivated) and a holding potential of -100 mV (both LVA and HVA currents activated). HVA currents were subtracted from HVA+LVA currents to yield the LVA current. Peak LVA currents were not different between hypertensive (8.9+/-0.8 pA/pF) and normotensive (7.8+/-0.6 pA/pF) groups of NTS neurons (P=0.27). These results demonstrate that 4 weeks of renal wrap hypertension induce an increase in Ca(2+) influx through HVA VGCCs in NTS neurons receiving arterial baroreceptor inputs.

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons.

Low voltage–activated (LVA) T-type Ca2+ (ICaT) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and ICaT currents. NaN/Nav1.9 was inhibited by inorganic ICa blockers as follows (IC50, μM): La3+ (46) > Cd2+ (233) > Ni2+ (892) and by mibefradil, a non-dihydropyridine ICaT antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni2+-resistant ICaT (AR-ICaT) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Aδ–like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aα/β cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-ICaT was restricted to CII-nociceptors, putative D-hair cells, and Aα/β-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aα/β-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.

T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

T-type channels are transient low-voltage-activated (LVA) Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarizations around resting potential. Studies in the past 20 years focused on the biophysical, physiological, and molecular characterization of T-type channels in most tissues. This led to a well-defined picture of the functional role of LVA channels in controlling low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, cell growth and differentiation. So far, little attention has been devoted to the role of T-type channels in transmitter release, which mainly involves channel types belonging to the high-voltage-activated (HVA) Ca(2+) channel family. However, evidence is accumulating in favor of a unique participation of T-type channels in fast transmitter release. Clear data are now reported in reciprocal synapses of the retina and olfactory bulb, synaptic contacts between primary afferent and second order nociceptive neurons, rhythmic inhibitory interneurons of invertebrates and clonal cell lines transfected with recombinant alpha(1) channel subunits. T-type channels also regulate the large dense-core vesicle release of neuroendocrine cells where Ca(2+) dependence, rate of vesicle release, and size of readily releasable pool appear comparable to those associated to HVA channels. This suggests that when sufficiently expressed and properly located near the release zones, T-type channels can trigger fast low-threshold secretion. In this study, we will review the main findings that assign a specific task to T-type channels in fast exocytosis, discussing their possible involvement in the control of the Ca(2+)-dependent processes regulating exocytosis like vesicle depletion and vesicle recycling.

Spike-Mediated and Graded Inhibitory Synaptic Transmission Between Leech Interneurons: Evidence for Shared Release Sites

Inhibitory synaptic transmission between leech heart interneurons consist of two components: graded, gated by Ca2+ entering by low-threshold [low-voltage-activated (LVA)] Ca channels and spike-mediated, gated by Ca2+ entering by high-threshold [high-voltage-activated (HVA)] Ca channels. Changes in presynaptic background Ca2+ produced by Ca2+ influx through LVA channels modulate spike-mediated transmission, suggesting LVA channels have access to release sites controlled by HVA channels. Here we explore whether spike-mediated and graded transmission can use the same release sites and thus how Ca2+ influx by HVA and LVA Ca channels might interact to evoke neurotransmitter release. We recorded pre- and postsynaptic currents from voltage-clamped heart interneurons bathed in 0 mM Na+/5 mM Ca2+ saline. Using different stimulating paradigms and inorganic Ca channel blockers, we show that strong graded synaptic transmission can occlude high-threshold/spike-mediated synaptic transmission when evoked simultaneously. Suppression of LVA Ca currents diminishes graded release and concomitantly increases the ability of Ca2+ entering by HVA channels to release transmitter. Uncaging of Ca chelator corroborates that graded release occludes spike-mediated transmission. Our results indicate that both graded and spike-mediated synaptic transmission depend on the same readily releasable pool of synaptic vesicles. Thus Ca2+, entering cells through different Ca channels (LVA and HVA), acts to gate release of the same synaptic vesicles. The data argue for a closer location of HVA Ca channels to release sites than LVA Ca channels. The results are summarized in a conceptual model of a heart interneuron release site.

Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-type Calcium Channels, from the Chinese Bird Spider Ornithoctonus huwena

Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with omega-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA approximately HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.

Transfer of β subunit regulation from high to low voltage-gated Ca 2+ channels

High voltage-activated Ca 2+ channel expression and gating is controlled by their β subunits. Although the sites of interaction are known at the atomic level, how β modulates gating remains to be determined. Using a chimeric approach, β subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the α 1 interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where β subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.

Expression of RNA of Subunits of Low-Threshold Calcium Channels in the Laterodorsal Nucleus of the Rat Thalamus: an Ontogenetic Aspect

It was shown earlier that low-threshold calcium current in neurons of the laterodorsal nucleus of the rat thalamus is of a multicomponent nature, and, moreover, its composition changes in the course of postnatal development of the animal. These findings raised a question on the ontogenetic development of expression of the pore-forming subunits of low voltage-activated (LVA) calcium channels (or T-type channels). At present, three isoforms of such subunits, α 1G, α1I, and α1H, have been cloned. In our study, we examined expression of mRNA of three subunits of T-type calcium channels in the laterodorsal thalamic nucleus of 1-, 5-, 15-, and 90-day (3-month)-old Wistar rats using reverse transcription and the polymerase chain reaction (PCR). Analysis of amplificates showed that mostly RNAs coding α1G and α1I subunits are expressed in the laterodorsal nucleus of rats of all age groups, while levels of expression RNAs of α1H and α1E subunits (the latter subunit of calcium channels of R type is close in its properties to subunits of LVA channels) are considerably lower (16 or 32 times smaller than the respective values for α1G and α1I subunits). We could not find a significant dependence of the expression of subunit RNAs of the above-mentioned calcium channels on the phases of ontogenetic development. It is possible to hypothesize that the age dynamics of the recorded calcium current in neurons of the laterodorsal thalamic nucleus is not related to increases in the transcription of the corresponding genes but results from post-translational changes in α1G and α1I subunits of proteins of LVA calcium channels or age-related peculiarities of regulation.

Ion Trafficking through T-type Ca2+ Channels

Voltage-gated Ca2+ channels play a key role in controlling Ca2+ entry during cell depolarization. At least 10 genes encode the main (α1) subunits of voltage-gated Ca2+ channels, which have been grouped into two main classes: the high voltage–activated (HVA) and the low voltage–activated (LVA) channels. HVA channels are primarily involved in muscle contraction, synaptic transmission, and hormone secretion, while LVA channels are associated with action potential generation and repetitive electrical activity. Structurally speaking, the Ca2+ channel α1 subunits forming the pore share strong similarities with other voltage-gated ion channels, in particular with Na+-conducting pores (Hille, 2001). Each α1 subunit has four domains (I–IV) linked together in a single polypeptide chain and each domain contains six putative transmembrane segments (S1–S6), plus a loop (P) that dips partially into the pore to, presumably, form the pore lining. Functionally speaking, the most striking similarity between Ca2+ and Na+ channels is represented by the LVA (T-type) channels, which have comparably low threshold for activation (−50 to −40 mV in 5 mM Ca2+) and inactivate fully and rapidly, if at a 20- to 40-fold lower rate than Na+ channels. Like Na+ channels, fast inactivation of T-type channels is strictly voltage rather than Ca2+ dependent, as in the case of channel types of the HVA family (L, N, P/Q, and R). T-type channels, however, possess other properties that are unique in comparison to other Ca2+ channels: (a) they deactivate more slowly (τdeact = 2.5 ms at −110 mV in 5 mM Ca2+; Carbone and Lux, 1984a); (b) they inactivate at relatively negative holding potentials; (c) they are equally permeable to Ca2+ and Ba2+; (d) they have small single channel conductance; and (e) they outlast membrane-patch excision since they do not require specific metabolic factors to preserve their activity (Carbone and Lux, 1984b, 1987).