Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.     

Decreased calcium accumulation in isolated nerve endings during hibernation in ground squirrels

Resting and depolarization-induced⁴⁵CaCl₂ accumulation was compared for synaptosomes isolated from hibernating and nonhibernating ground squirrels. Channel subtype antagonists were used to identify the active voltage-sensitive calcium channel subtypes in these preparations. There was significantly less⁴⁵Ca²⁺ accumulation in synaptosomes isolated from hibernating as compared to cold-adapted nonhibernating ground squirrels in both basal (p<0.005) and depolarizing (p<0.03) media over a 30 sec to 5 min incubation period. The elevation in⁴⁵Ca²⁺ accumulation triggered by K⁺ depolarization was blocked by 50 μM CdCl₂, 1 μM ω-conotoxin MVIIC or 1 μM ω-agatoxin IVA. Inhibition was not observed with 1 μM nifedipine or with 1 μM ω-conotoxin GVIA. These results suggest that hibernation is associated with reduced presynaptic⁴⁵Ca²⁺ conductance via voltage-sensitive channels with a pharmacological sensitivity that is different from the established L-, N-, and P-types in other systems but share features of the recently described Q-type calcium channel. This decrease may reflect a cellular adaptation that helps confer tolerance to the near total cerebral ischemia associated with hibernation.     

Role of N‐ and L‐type calcium channels in depolarization‐induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals

Multiple types of voltage‐activated calcium (Ca²⁺) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca²⁺ influx through N‐ and L‐ type voltage‐activated Ca²⁺ channels in sympathetic neurons to the depolarization‐induced activation of tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine (NE) synthesis, and the depolarization‐induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both ω‐conotoxin GVIA (1 μM), a specific blocker of N‐type channels, and nimodipine (1 μM), a specific blocker of L‐type Ca²⁺ channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K⁺, indicating that Ca²⁺ influx through both types of channels contributes to enzyme activation. In contrast, K⁺ stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by ω‐conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K⁺ stimulation of NE release from both ganglia and irises was also blocked completely when ω‐conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca²⁺ influx through N‐ and L‐type Ca²⁺ channels. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 137–148, 1999     

Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels

The antibodies against omega-conotoxin GVIA (-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B₁Nt (N-terminal segment [residues 1–13] of BI ₁ subunits of VDCCs) were prepared, and the selectivity for each antigen -CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–-CTX GVIA antibody against -CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM -CTX GVIA, but not by -CTX SVIB (N-type VDCC blocker), -CTX MVIIC (N- and P-type VDCC blocker), or -Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC₅₀ values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 M B1Nt. These results suggest that the antibodies prepared against -CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against -CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti -CTX GVIA antibody must also be useful for the radioimmunoassay of -CTX GVIA.     

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.     

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel

A novel series of substituted tetrahydropyrrolo[3,4-c]pyrazoles were investigated as blockers of the N-type calcium channel (Ca_v2.2 channels), a chronic pain target.     

Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.     

N型钙通道与疼痛

N型电压依赖性钙通道(VDCCs)在疼痛的传递与调控中具有重要作用。它们密集分布于脊髓背角伤害感受性神经元突触前末梢,参与主要疼痛介质如谷氨酸和P物质等释放的调节。通过阻断上述通道,选择性N型VDCCs阻断剂表现出强效镇痛作用,N型VDCCs Cav2．2亚基基因敲除小鼠也表现为痛阈提高。N型VDCCs还分布于自主神经系统和中枢神经系统突触部位,现有的N型VDCCs阻断剂用于疼痛治疗时出现的各种副作用与这些部位的突触抑制有关。最近发现,背根节伤害感受性神经元上存在一种特异的N型VDCCs亚型,这为疼痛治疗提供了一个非常有意义的新靶标。     

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Ca_v2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.     

A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.

A common structural motif consisting of a cystine knot and a small triple-stranded beta-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides omega-conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI-I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes. The other component of this motif is a triple-stranded, anti-parallel beta-sheet containing a minimum of 10 residues, XXC2, XC5X, XXC6X (where the numbers on the half-cysteine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cysteine knot and antiparallel beta-sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

Scaffold-based design and synthesis of potent N-type calcium channel blockers

The therapeutic agents flunarizine and lomerizine exhibit inhibitory activities against a variety of ion channels and neurotransmitter receptors. We have optimized their scaffolds to obtain more selective N-type calcium channel blockers. During this optimization, we discovered NP118809 and NP078585, two potent N-type calcium channel blockers which have good selectivity over L-type calcium channels. Upon intraperitoneal administration both compounds exhibit analgesic activity in a rodent model of inflammatory pain. NP118809 further exhibits a number of favorable preclinical characteristics as they relate to overall pharmacokinetics and minimal off-target activity including the hERG potassium channel.     

Aldosterone regulation of T-type calcium channels

Voltage-operated calcium channels play a crucial role in signal transduction in many excitable and non-excitable cell types. While a rapid modulation of their activity by hormone-activated kinases and/or G proteins has been recognized for a long time, a sustained control of their expression level has been only recently demonstrated. In adrenal H295R cells, for example, aldosterone treatment selectively increased low threshold T-type calcium current density without affecting L-type currents. Antagonizing the mineralocorticoid receptor (MR) with spironolactone prevented aldosterone action on T-type currents. By RT-PCR, we detected in these cells the presence of two different isoforms of L-type channels, alpha(1)C and alpha(1)D, and one isoform of T channel, alpha(1)H. A second T channel isoform (alpha(1)G) was also observed under particular culture conditions. Quantification of the specific messenger RNA by real time RT-PCR allowed us to show a 40% increase of the alpha1H messenger levels upon aldosterone treatment (alpha(1)G was insensitive), a response that was also completely prevented by spironolactone. Because T-type, but not L-type channel activity is linked to steroidogenesis, this modulation represents a positive, intracrine feed back mechanism exerted by aldosterone on its own production.Aldosterone has been also implicated in the pathogenesis and progression of ventricular hypertrophy and heart failure independently of its action on arterial blood pressure. We have observed that, in rat neonatal cardiomyocytes, aldosterone increases (by two-fold) L-type calcium current amplitude in ventricular but not in atrial cells. No significant effect of aldosterone could be detected on T-type currents, that were much smaller than L-type currents in these cells. However, aldosterone exerted opposite effects on T channel isoform expression, increasing alpha(1)H and decreasing alpha(1)G. Although the functional role of T channels is still poorly defined in ventricular cardiomyocytes, an overexpression of alpha(1)H could be partially responsible for the arrhythmias linked to hyperaldosteronism.Finally, T channels also appear to be involved in the neuroendocrine differentiation of prostate epithelial cells, a poor prognosis in prostate cancer. We have shown that the only calcium channel expressed in the prostatic LNCaP cells is the alpha(1)H isoform and that induction of cell differentiation with cAMP leads to a concomitant increase in both T-type current and alpha(1)H mRNA. In spite of the presence of MR in these cells, aldosterone only modestly increased alpha(1)H mRNA levels. A functional role for these channels was suggested by the observation that low nickel concentrations prevent neuritic process outgrowth.In conclusion, it appears that T-type calcium channel expression vary in different patho-physiological conditions and that aldosterone, in several cell types, is able to modulate this expression.     

Molecular determinants of Rem2 regulation of N-type calcium channels

Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel β subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca_v β subunit and to regulate the activities of Ca_v2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca_v β₃ interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the β subunit were able to reduce whole cell calcium currents.     

The neuronal calcium ion channel activity of constrained analogues of MONIRO-1

Structural modifications of the neuronal calcium channel blocker MONIRO-1, including constraining the phenoxyaniline portion of the molecule and replacing the guanidinium functionality with tertiary amines, led to compounds with significantly improved affinities for the endogenously expressed Ca_V2.2 channel in the SH-SY5Y neuroblastoma cell line. These analogues also showed promising activity towards the Ca_V3.2 channel, recombinantly expressed in HEK293T cells. Both of these ion channels have received attention as likely targets for the treatment of neuropathic pain. The dibenzoazepine and dihydrobenzodiazepine derivatives prepared in this study show an encouraging combination of neuronal calcium ion channel inhibitory potency, plasma stability and potential to cross the blood–brain-barrier.     

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.     

芋螺毒素与钙离子通道相互作用的计算机模拟

电压门控N－型钙离子通道是与神经元中释放的神经信号传递有关的跨细胞膜的特殊蛋白质分子,它由好几个蛋白质亚基组成,其中的α1亚基包含了电压敏感器和钙离子的选择性孔道,该亚基的一级结构已经发表,一般认为α1亚基包含4个重复单位（I－Ⅳ）,每个重复单位包括6段跨膜区（S1－S6）,其中跨膜区S4上有很多正电荷,被认为是通道的电压敏感器,S5和S6之间的连接区（P区）被认为是形成通道的门孔的部分,N－型钙离子通道能够被一些w-芋螺毒素特异性在阻断,这些ω－芋螺毒素的三维结构已经由二维核磁共振方法测定,尽管还没有被证实,但一般认为w-芋螺毒素占据了通道的孔道,有实验证明,钙离子第三个重复单位的P区（ⅢP区）是通道的芋螺毒素结合的主要部位,在本中,我们用分子模拟程序建模了ⅢP区的结构,为了通道的阻断机理有一个清楚的了解,我们利用分子对接程序模拟了IIIP区和三种芋螺毒素GVA,MVIIA和S03作用的理论模型,在我们的模型中,GVIA与钙通道的作用方式可能与MVIIA不同,而M VII A和SO3与钙通道的作用方式可能相同,我们还讨论了这些芋螺毒素中的关键残基的作用。     

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca²⁺ leak using synthetic Ca²⁺ indicators that report changes in both the cytoplasmic ([Ca²⁺]_i) and the luminal ER ([Ca²⁺]_ER) Ca²⁺ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca²⁺ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca²⁺]_ER but did not decrease the ER Ca²⁺ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP₃R and only Orai3 channel supported the 2-APB-induced ER Ca²⁺ leak, while Orai1 and Orai2 inhibited this type of ER Ca²⁺ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca²⁺ leak but did not modify the ER Ca²⁺ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca²⁺]_i response after TG but only when the ER store had been overloaded with Ca²⁺ by eliminating the acidic internal Ca²⁺ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca²⁺ leak but forms an ER Ca²⁺ leak channel that is limiting the overloading with Ca²⁺ of the ER store.