Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific¹²⁵I-omega-conotoxin (-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited¹²⁵I--CgTX binding, but not that of [³H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of¹²⁵I--CgTX, because the venom also inhibited the binding of [³H](+)PN200-110 to a similar degree. The amount of specific binding of¹²⁵I--CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with¹²⁵I--CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of -CgTX-sensitive Ca channels in crude membranes from rat whole brain and that -CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω-conotoxin-GVIA, ω-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.     

Binding and labeling of omega-conotoxin GVIA in crude membranes from subfractionated fractions and various areas of chick brain

Specific binding and specific labeling of¹²⁵I-ω-CgTX were investigated in crude membranes from both subfractionated fractions and various brain areas in chick whole brain. The specific activities of the marker enzymes 2′,3′-cyclic nucleotide 3′-phosphorylase, Na/K ATPase and succinic dehydrogenase in the subfractionated fractions were three- to five-fold higher than those in the P₂ fraction. However, the amount of specific [¹²⁵I]ω-CgTX binding in the fractions of synaptosomes and synaptic plasma membranes was only about 1.2-times higher than that in the P₂ fraction. The characteristics of specific¹²⁵I-ω-CgTX labeling with disccinimidyl suberate to the 135-kDa band were generally comparable to those of specific [¹²⁵I]ω-CgTX binding sites. These results suggest that the specific binding sites of [¹²⁵I]ω-CgTX were not localized the synaptosomes and synaptic plasma membranes fractions, although each fraction was well isolated from the others from which were decided by the strength of specific activity for marker enzymes.     

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.     

Inhibition of Ischemia-Induced Dopamine Release by ω-Conotoxin, a Calcium Channel Blocker, in the Striatum of Spontaneously Hypertensive Rats: In Vivo Brain Dialysis Study

The effect of omega-conotoxin GVIA (CgTX), an N-and L-type voltage-sensitive calcium channel (VSCC) blocker, on the release of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum before and during transient cerebral ischemia in spontaneously hypertensive rats was studied using an in vivo brain dialysis technique. Continuous perfusion of CgTX in the striatum was started 20 min before ischemia and concentrations of dopamine and DOPAC in the dialysate were measured using HPLC with an electro-chemical detector. Before ischemia, both 10 and 100 microM CgTX significantly lowered the concentration of dopamine, to 49% of the basal values. DOPAC concentrations also decreased significantly, by 28 and 17%, respectively. Forebrain ischemia, produced by bilateral carotid artery occlusion, reduced striatal blood flow to less than 6% of the resting value in each group. During 20 min of ischemia, the vehicle group showed a marked increase in dopamine (175 times the basal concentration). In the 10 or 100 microM CgTX perfusion group, in contrast, dopamine release was significantly attenuated, to 38 or 29% of the vehicle group, respectively. DOPAC concentrations decreased during ischemia to 58% of the basal value in the vehicle group and 49% in both CgTX groups. These results indicate that the massive release of striatal dopamine during ischemia depends largely on the influx of extracellular calcium via CgTX-sensitive VSCCs.     

Solution structure of alpha-conotoxin PIA, a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors

alpha-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing alpha6 and alpha3 subunits. alpha-conotoxin PIA displays 75-fold higher affinity for rat alpha6/alpha3beta2beta3 nAChRs than for rat alpha3beta2 nAChRs. We have determined the three-dimensional structure of alpha-conotoxin PIA by nuclear magnetic resonance spectroscopy. The alpha-conotoxin PIA has an "omega-shaped" overall topology as other alpha4/7 subfamily conotoxins. Yet, unlike other neuronally targeted alpha4/7-conotoxins, its N-terminal tail Arg1-Asp2-Pro3 protrudes out of its main molecular body because Asp2-Pro3-Cys4-Cys5 forms a stable type I beta-turn. In addition, a kink introduced by Pro15 in the second loop of this toxin provides a distinct steric and electrostatic environment from those in alpha-conotoxins MII and GIC. By comparing the structure of alpha-conotoxin PIA with other functionally related alpha-conotoxins we suggest structural features in alpha-conotoxin PIA that may be associated with its unique receptor recognition profile.     

Development of antibody-based assays for omega-conotoxin MVIIA

Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.