N-type Calcium Channel Activity Research Articles

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5-bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

Sympathetic nerves regulate vascular tone by releasing neurotransmitters into the vasculature. We previously demonstrated that bradykinin facilitates sympathetic neurotransmission in rat mesenteric arteries. Although little is known about the intracellular mechanism modulating this neurotransmission, recent cell line experiments have shown that the KCNQ channel, which is inhibited by the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP₂), participates in the control of neurotransmission by bradykinin. In the present study, we examined the mechanism regulating neurotransmitter release from rat perivascular sympathetic nerves. Excitatory junction potentials (EJPs) elicited by repetitive nerve stimulation (1 Hz, 11 pulses, 20 μs, 20-50 V), a measure of sympathetic purinergic neurotransmission, were recorded with a conventional microelectrode technique in rat mesenteric arteries. Bradykinin (10⁻⁷ mol l⁻¹) significantly enhanced the amplitude of EJPs (n=22, P<0.05). This enhancing effect was abolished by N-type calcium-channel inhibition with ω-conotoxin GVIA (2 × 10⁻⁹ mol ⁻¹l, n=8). The blockade of phospholipase C with U-73122 (10(-6) mol l⁻¹, n=17) also eliminated the facilitatory effect of bradykinin. In addition, the effects of bradykinin were diminished by the prevention of PIP₂ resynthesis with wortmannin (10⁻⁵ mol l⁻¹ n=7) or KCNQ channel inhibition with XE-991 (10⁻⁵ mol l⁻¹, n=7). On the other hand, depletion of intracellular calcium stores with cyclopiazonic acid (3 × 10⁻⁶ mol l⁻¹, n=6) or the inhibition of protein kinase C with bisindolylmaleimide-I (10⁻⁶ mol l⁻¹, n=9) did not alter the action of bradykinin. These data demonstrate that the hydrolysis of PIP₂ by phospholipase C, which is activated by G(q/11)-coupled receptors, and subsequent KCNQ channel inhibition enhance sympathetic purinergic neurotransmission presumably via the activation of N-type calcium channels in rat mesenteric arteries.

Psychiatric Predisposition to Autonomic and Abnormal Perception Side-Effects of Ziconotide: A Case Series Study

Ziconotide is a reversible blocker of the N-type neuronal voltage-sensitive calcium channels with analgesic effects. The main adverse effects of ziconotide are ataxia, dizziness, gait disorder, confusion, hallucinations, and gastrointestinal symptoms. Eighteen chronic pain patients with intrathecal ziconotide treatment were investigated using the Mini International Neuropsychiatric Interview for psychiatric disorders according to the DSM IV. Ten patients showed good pain relief (ΔVAS ≥ 50%) after one year of treatment. Patients without psychiatric comorbidity exhibited better outcomes, without autonomic side-effects. Eight patients with panic disorder (always comorbid with other psychiatric disorders) showed the greatest number of side-effects during treatment with ziconotide. Emotional and cognitive symptoms of panic disorder are associated with autonomic symptoms resulting from parasympathetic activation. Dysfunction of both cholinergic and N-type calcium channel activity was found. A psychiatric disorder with cholinergic-noradrenergic system impairment could increase some side-effects of treatment with N-type calcium channel blockers.

A Nonadherent Cell-Based HTS Assay for N-Type Calcium Channel Using Calcium 3 Dye

The N-type calcium channel is a member of the voltage-sensitive calcium channel family and plays a major role in the regulation of neurotransmitter release in the central and peripheral nervous systems. Inhibition of the N-type calcium channel by intrathecal administration of the channel-specific blocker omega-conotoxin MVIIA (ziconotide) is efficacious in the treatment of severe chronic pain. While no orally active small molecules that block the N-type calcium channel are currently available, the discovery of such potentially valuable therapeutics would benefit from a reliable, high throughput assay. However, the assay of N-type calcium channel activity by measuring calcium influx using nonadherent cells in a high throughput fashion has not been achieved before, likely owing to a number of technical hurdles. For example, the measurement of calcium levels in nonadherent cells using conventional calcium indicators, such as Fluo-3 or Fluo-4, requires dyeloading the cells in suspension and subsequent removal of extracellular dye. This limits plate throughput and requires constant handling of the cells. To assay the N-type calcium channel activity using a nonadherent cell line in a high throughput manner, we investigated the application of no-wash calcium assay kits from Molecular Devices Corp. (Sunnyvale, CA): FLIPR Calcium, FLIPR Calcium Plus, and FLIPR Calcium 3. We show here that the FLIPR Calcium 3 assay kit can be used with nonadherent IMR-32 cells to measure potassium-evoked, omega-conotoxin MVIIA-reversible calcium flux with high throughput (15,000 data points/day), high quality (Z approximately 0.6), and minimal handling of the cells. Thus, this assay can be used to reliably and efficiently screen large compound libraries in the search for small molecule N-type calcium channel blockers.

Contribution of the kinetics of G protein dissociation to the characteristic modifications of N-type calcium channel activity

Direct G protein inhibition of N-type calcium channels is recognized by characteristic biophysical modifications. In this study, we quantify and simulate the importance of G protein dissociation on the phenotype of G protein-regulated whole-cell currents. Based on the observation that the voltage-dependence of the time constant of recovery from G protein inhibition is correlated with the voltage-dependence of channel opening, we depict all G protein effects by a simple kinetic scheme. All landmark modifications in calcium currents, except inhibition, can be successfully described using three simple biophysical parameters (extent of block, extent of recovery, and time constant of recovery). Modifications of these parameters by auxiliary β subunits are at the origin of differences in N-type channel regulation by G proteins. The simulation data illustrate that channel reluctance can occur as the result of an experimental bias linked to the variable extent of G protein dissociation when peak currents are measured at various membrane potentials. To produce alterations in channel kinetics, the two most important parameters are the extents of initial block and recovery. These data emphasize the contribution of the degree and kinetics of G protein dissociation in the modification of N-type currents.

Striatal metabotropic glutamate receptors as a target for pharmacotherapy in Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopamine (DA)-containing neurons in the substantia nigra pars compacta (SNc). The symptoms are resting tremor, slowness of movement, rigidity and postural instability. Evidence that an imbalance between dopaminergic and cholinergic transmission takes place within the striatum led to the utilization of DA precursors, DA receptor agonists and anticholinergic drugs in the symptomatic therapy of PD. However, upon disease progression the therapy becomes less effective and debilitating effects such as dyskinesias and motor fluctuations appear. Hence, the need for the development of alternative therapeutic strategies has emerged. Several observations in different experimental models of PD suggest that blockade of excitatory amino acid transmission exerts antiparkinsonian effects. In particular, recent studies have focused on metabotropic glutamate receptors (mGluRs). Drugs acting on group I and II mGluRs have indeed been proven useful in ameliorating the parkinsonian symptoms in animal models of PD and therefore might represent promising therapeutic targets. This beneficial effect could be due to the reduction of both glutamatergic and cholinergic transmission. A novel target for drugs acting on mGluRs in PD therapy might be represented by striatal cholinergic interneurons. Indeed, the activation of mGluR2, highly expressed on this cell type, is able to reduce calcium-dependent plateau potentials by interfering with somato-dendritic N-type calcium channel activity, in turn reducing ACh release in the striatum. Similarly, the blockade of both group I mGluR subtypes reduces cholinergic interneuron excitability, and decreases striatal ACh release. Thus, targeting mGluRs located onto cholinergic interneurons might result in a beneficial pharmacological effect in the parkinsonian state.

A model for a G-protein-mediated mechanism for synaptic channel modulation

Neurons communicate with other neurons via specialized structures called synapses, at which the digital voltage signal encoded in an action potential is converted into an analog chemical signal. An action potential that arrives at the presynaptic face triggers release of neurotransmitter from vesicles in a calcium-dependent manner, and the neurotransmitter diffuses across the synaptic cleft and binds to receptors on the post-synaptic face, where it may trigger a postsynaptic action potential. Calcium is a critical component of the release process, and its spatio-temporal dynamics can control the release and can lead to facilitation or augmentation. However, how cells regulate cytoplasmic calcium so that exocytosis can be triggered successfully is still not completely understood. We propose a mechanism, based upon the experimental findings of Barrett and Rittenhouse [C.F. Barrett, A.R. Rittenhouse, Modulation of N-type calcium channel activity by G-proteins and protein kinase C, J. Gen. Physiol. 115 (3) (2000) 277], for the regulation of calcium influx through N-type channels in the presynaptic terminal by PKC and downstream effectors of G-protein activation. This proposed modulatory mechanism consists of a feedback loop involving cytoplasmic calcium, neurotransmitters and G-protein-coupled receptors. We study the dynamics of each component separately and then we address how kinetic properties of the components and the frequency of the stimuli affect the regulatory mechanisms presented here.

Caveolin-1 Expression and Membrane Cholesterol Content Modulate N-Type Calcium Channel Activity in NG108-15 Cells

Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca 2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma × glioma NG108-15 hybrid cells [cav1(+) clone] that lack endogenous caveolins but express N-type Ca 2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells demonstrated that N-type current density was reduced in size by ∼70% without any significant change in the time course of activation and inactivation and voltage dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild-type NG108-15 cells. To gain insight into the mechanism underlying caveolin-1 lowering of N-current density, and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild-type NG108-15 cells enriched with cholesterol after exposure to a methyl- β-cyclodextrin-cholesterol complex. A lower Ca 2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps.

Calcium Channel and NMDA Receptor Activities Differentially Regulate Nuclear C/EBPβ Levels to Control Neuronal Survival

Insulin-like growth factor-1 (IGF-1) promotes the survival of cerebellar granule neurons by enhancing calcium influx through L-type calcium channels, whereas NMDA receptor-mediated calcium influx can lead to excitotoxic death. Here we demonstrate that L and NMDA receptor channel activities differentially regulate the transcription factor C/EBPβ to control neuronal survival. Specifically, we show that L channel-dependent calcium influx results in increased CaMKIV activity, which acts to decrease nuclear C/EBPβ levels. Conversely, NMDA receptor-mediated influx rapidly elevates nuclear C/EBPβ and induces excitotoxic death via activation of the calcium-dependent phosphatase, calcineurin. Moderate levels of AMPA receptor activity stimulate L channels to improve survival, whereas higher levels stimulate NMDA receptors and reduce neuronal survival, suggesting differential synaptic effects. Finally, N-type calcium channel activity reduces survival, potentially by increasing glutamate release. Together, these results show that the L-type calcium channel-dependent survival and NMDA receptor death pathways converge to regulate nuclear C/EBPβ levels, which appears to be pivotal in these mechanisms.

Molecular determinants of cysteine string protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Galpha subunits but not Galphabeta subunits whereas the C terminal binding site of CSP associates with either free Galphabeta subunits or Galphabeta in complex with Galpha. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.

Modulation of N-type calcium channel activity by G-proteins and protein kinase C.

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.

Role of disulfide bridges in the folding, structure and biological activity of ω-conotoxin GVIA

ω-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by 1H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379–384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in ω-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.

Roles of key functional groups in omega-conotoxin GVIA synthesis, structure and functional assay of selected peptide analogues.

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 12014-12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d-Tyr and the d-form and l-form of the constrained analogue 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of omega-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and omega-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.

PGE2 increases substance P release from renal pelvic sensory nerves via activation of N-type calcium channels.

Activation of renal pelvic sensory nerves by increased pelvic pressure results in a renal pelvic release of substance P that is dependent on intact prostaglandin synthesis. An isolated renal pelvic wall preparation was used to examine whether PGE2 increases the release of substance P from renal pelvic sensory nerves and by what mechanisms. The validity of the model was tested by examining whether 50 mM KCl increased substance P release from the pelvic wall. Fifty millimolar KCl produced an increase in substance P release, from 9.6 +/- 1.6 to 26.8 +/- 4.0 pg/min, P < 0.01, that was blocked by the L-type calcium blocker verapamil (10 microM). PGE2 (0.14 microM) increased the release of substance P from the pelvic wall from 8.9 +/- 0.9 to 20.6 +/- 3.3 pg/min, P < 0.01. PGE2 failed to increase substance P release in a calcium-free medium. The PGE2-induced substance P release was blocked by the N-type calcium blocker omega-conotoxin (0.1 microM) but was unaffected by verapamil. In conclusion, PGE2 increases the release of substance P from renal pelvic sensory nerves by a calcium-dependent mechanism that requires influx of calcium via N-type calcium channels.

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen.

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.

Fluspirilene block of N‐type calcium current in NGF‐differentiated PC 12 cells

1. High voltage-activated calcium currents were recorded in nerve growth factor (NGF)-differentiated PC12 cells with the whole-cell patch clamp technique. After exposure to NGF for 3-10 days the PC12 cells developed neurone-like processes and calcium currents which were pharmacologically separable into L- and N-types (defined by sensitivity to nifedipine and omega-conotoxin GVIA respectively). 2. After blocking the L-type calcium channels with nifedipine (10 microM), omega-conotoxin GVIA blocked approximately 85% of the remaining calcium current with an IC50 of 3 nM and a Hill coefficient of 1. The block by conotoxin GVIA was irreversible on the time scale of these experiments. These results suggested that the majority of the nifedipine-insensitive calcium current was N-type. 3. Fluspirilene, a substituted diphenylbutylpiperidine with potent neuroleptic properties, reversibly inhibited the N-type component in a dose-dependent manner with an IC50 of 30 nM. The Hill coefficient of the block was 0.25. The fraction of current blocked was the same at all test potentials examined (-30 to +40 mV). 4. These data indicate that the neuroleptic properties of fluspirilene may be due, at least in part, to an inhibition of neuronal N-type calcium channels. This finding raises the possibility that modulation of N-type calcium channel activity by drugs derived from substituted diphenylbutylpiperidines may provide a novel way of altering neurotransmitter release and hence brain function.

Multiple modes of N-type calcium channel activity distinguished by differences in gating kinetics

In many neurons, N-type Ca2+ channels are a major Ca2+ entry pathway and strongly influence neurotransmitter release. We carried out cell-attached patch recordings (110 mM Ba2+ as charge carrier) to characterize the rapid opening and closing kinetics of N-type Ca2+ channel gating in frog sympathetic neurons. Single channels display at least three distinct patterns of gating, characterized as low-, medium-, and high-rho o modes on the basis of channel open probability (rho o) during depolarizing pulses to -10 mV. Spontaneous transitions from one mode to another are infrequent, with an exponential distribution of dwell times and mean sojourns of approximately 10 sec in each mode. Thus, a channel typically undergoes hundreds or thousands of open-closed transitions in one mode before switching to a different mode. Transitions between modes during a depolarization were occasionally detected, but were rare, as expected for infrequent modal switching. Within each mode, the activation kinetics were well described by a simple scheme (C2-C1-O), as previously reported for other types of Ca2+ channels. Rate constants are strikingly different from one mode to another, giving each mode its own characteristic kinetic signature. The gating behavior at -10 mV ranges from brief openings (approximately 0.3 msec) and long closures (10-20 msec) for low-rho o gating to long openings (3 msec) and brief closures (approximately 1 msec) for high-rho o gating. Intermediate values for mean open durations (approximately 1.5 msec) and mean closed durations (approximately 3 msec) were found for medium-rho o gating. In addition to being kinetically distinct, channel openings in the low-rho o mode often exhibit a unitary current approximately 0.2 pA larger than in the medium- or high-rho o mode. Each mode is characterized by its own voltage dependence: activation occurs at relatively negative potentials and is most steeply voltage dependent in the high-rho o mode, while activation requires very strong depolarizations and is weakly voltage dependent in the low-rho o mode. The proportion of time spent in the individual modes varies greatly from one patch to another, suggesting that modal gating may be subject to cellular control.

Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel.

N-type calcium channels are omega-conotoxin (omega-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the alpha 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel alpha 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (alpha 1B-1 and alpha 1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The alpha 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal beta 2 and alpha 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by omega-CgTx but was insensitive to dihydropyridines. The alpha 1B-1 alpha 2b beta 2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) omega-CgTx binding sites. Co-expression of the beta 2 subunit was necessary for N-type channel activity, whereas the alpha 2b subunit appeared to modulate the expression of the channel. The heterogeneity of alpha 1B subunits, along with the heterogeneity of alpha 2 and beta subunits, is consistent with multiple, biophysically distinct N-type calcium channels.

P-364 - Activation of N-type calcium channels by stannous chloride at motor nerve terminals.

P-364 - Activation of N-type calcium channels by stannous chloride at motor nerve terminals.