Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, α_1B and β₃ subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional ω-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of α_1B subunits was diffuse. The distribution of the α_1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 198–208, 1998     

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.     

Solution structure of ω-conotoxin MVIIA using 2D NMR spectroscopy

The solution structure of ω-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded β-sheet involving residues 6–8, 20–21, and 24–25. The structure of this toxin is very similar to that of ω-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.     

Refined solution structure of omega-conotoxin GVIA: implications for calcium channel binding.

The polypeptide omega-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.     

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.     

Successful reduction of off-target hERG toxicity by structural modification of a T-type calcium channel blocker

To obtain an optimized T-type calcium channel blocker with reduced off-target hERG toxicity, we modified the structure of the original compound by introducing a zwitterion and reducing the basicity of the nitrogen. Among the structurally modified compounds we designed, compounds 5 and 6, which incorporate amides in place of the original compound’s amines, most appreciably alleviated hERG toxicity while maintaining T-type calcium channel blocking activity. Notably, the benzimidazole amide 5 selectively blocked T-type calcium channels without inhibiting hERG (hERG/T-type ⩾ 220) and L-type channels (L-type/T-type = 96), and exhibited an excellent pharmacokinetic profile in rats.     

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.     

Stereochemical quality of protein structure coordinates.

Methods have been developed to assess the stereochemical quality of any protein structure both globally and locally using various criteria. Several parameters can be derived from the coordinates of a given structure. Global parameters include the distribution of phi, psi and chi 1 torsion angles, and hydrogen bond energies. There are clear correlations between these parameters and resolution; as the resolution improves, the distribution of the parameters becomes more clustered. These features show a broad distribution about ideal values derived from high-resolution structures. Some structures have tightly clustered distributions even at relatively low resolutions, while others show abnormal scatter though the data go to high resolution. Additional indicators of local irregularity include proline phi angles, peptide bond planarities, disulfide bond lengths, and their chi 3 torsion angles. These stereochemical parameters have been used to generate measures of stereochemical quality which provide a simple guide as to the reliability of a structure, in addition to the most important measures, resolution and R-factor. The parameters used in this evaluation are not novel, and are easily calculated from structure coordinates. A program suite is currently being developed which will quickly check a given structure, highlighting unusual stereochemistry and possible errors.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Normalization of deranged signal transduction in lymphocytes of COPD patients by the novel calcium channel blocker H-DHPM

Investigations on the role of intracellular Ca²⁺ ion concentration in the mechanism of development of COPD in smokers and non-smokers were carried out. The intracellular Ca²⁺ levels were found to be increased in human lymphocytes in patients with COPD as compared to non-smokers and smokers without COPD. The investigations reveal an association in altered intracellular Ca²⁺ regulation in lymphocytes and severity of COPD, by means of significant activation of Protein kinase C and inducible nitric oxide synthase (iNOS). The effect of a novel calcium channel blocker ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) as a potential candidate for the treatment of COPD was also investigated. H-DHPM treated cells showed a decrease in intracellular Ca²⁺ level as compared to the control cells. Molecular studies were carried out to evaluate the expression profile of NOS isoforms in human lymphocytes and it was shown that H-DHPM decreases the increased iNOS in COPD along with reestablishing the normal levels of endothelial nitric oxide synthase (eNOS). The results of H-DHPM were comparable with those of Amlodipine, a known calcium channel blocker. Calcium channel blocker H-DHPM proves to be a potential candidate for the treatment of COPD and further clinical studies are required to prove its role in the treatment of pulmonary hypertension (PH).     

Design and synthesis of novel anti-hyperalgesic agents based on 6-prenylnaringenin as the T-type calcium channel blockers

Since 6-prenylnaringenin (6-PNG) was recently identified as a novel T-type calcium channel blocker with the IC₅₀ value around 1?µM, a series of flavanone derivatives were designed, synthesized and subsequently evaluated for T-channel-blocking activity in HEK293 cells transfected with Ca_v3.2?T-type channels using a patch-clamp technique. As a result, several new flavanones blocked Ca_v3.2-dependent T-currents more potently than 6-PNG. In the synthesized compounds, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(2-hydroxyphenyl)chroman-4-one 8j, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11b, 6-(2-cyclopentylideneethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11d, and 6-(2-Cyclopentylethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 12c were more potent blocker than 6-PNG with the IC₅₀ value of 0.39, 0.26, 0.46, and 0.50?µM, respectively. Among the above four derivatives, the compound 8j provided the best result in the in vivo experiments; i.e. systemic administration of 8j at the minimum dose completely restored neuropathic pain induced by partial sciatic nerve ligation in mice.     

Pranidipine,a new 1, 4-dihydropyridine calcium channel blocker,enhances cyclic GMP-independent nitric oxide-induced relaxation of the rat aorta

Pranidipine, a new calcium channel modulator, prolonged endothelium-dependent relaxation induced by acetylcholine in a aortic ring preparation, contracted with prostaglandin F2. This action was not shared by amlodipine. The effect was not modified by indomethacin, suggesting that the action of pranidipine does not involve prostanoid metabolism. N^G-nitro-L-arginine completely prevented the action of Pranidipine. The drug affected neither nitric oxide (NO) synthase activity nor the level of cyclic GMP in the vessel. Pranidipine did not affect the sensitivity of the contractile proteins to calcium. Pranidipine also did not alter cyclic GMP-induced relaxation in a-toxinskinned vascular preparations. Pranidipine also prolonged glyceryl trinitrate-induced relaxation in the endothelium denuded rat aorta. Furthermore, pranidipine enhanced relaxation of the aorta induced by glyceryl trinitrate even in the presence of methylene blue, a guanylyl cyclase inhibitor. This action was not modified by iberiotoxin or by charybdotoxin, two inhibitors of the calciumactivated potassium channel. The results strongly suggest that pranidipine enhances cyclic GMPindependent NO-induced relaxation of smooth muscle by a mechanism other than through NOinduced hyperpolarization. These effects were in direct contrast to amlodipine, another new 1,4dihydropyridine calcium antagonist.     

T-type Ca2+ channel blockers suppress the growth of human cancer cells

In order to further clarify the role of T-type Ca²⁺ channels in cell proliferation, we have measured the growth inhibition of human cancer cells by using our potent T-type Ca²⁺ channel blockers. As a result, KYS05090, a most potent T-type Ca²⁺ channel blocker, was found to be as potent as doxorubicin against some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that T-type calcium channel is important in regulating the important cellular phenotype transition leading to cell proliferation, and thus novel T-type Ca²⁺ channel blocker presents new prospects for cancer treatment.     

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     

Separation of the optical isomers of a new 1,4-dihydropyridine calcium channel blocker (LF 2.0254) by liquid and supercritical fluid chromatography

The four optical isomers of a new calcium channel blocker LF 2.0254 (developed by Laboratoires Fournier) are resolved by chromatography using chiral stationary phases. Two methods are proposed: the first one involving two chiral stationary phases [(S)-N-(2-naphthyl)alanine, a Pirkle type CSP and Chiralcel OJ, a cellulose derived CSP] in the liquid chromatographic mode, the second one involving a single CSP (Chiralcel OJ) in the supercritical fluid chromatographic mode. © 1994 Wiley-Liss, Inc.