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.     

Monoclonal antibodies against the presynaptic calcium channel antagonist omega-conotoxin GVI A from cone snail poison

Monoclonal antibodies have been prepared against omega-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic omega-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block omega-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block omega-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom

The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.     

Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.     

Recordings from single neocortical nerve terminals reveal a nonselective cation channel activated by decreases in extracellular calcium

Synaptic activity causes reductions in cleft [Ca(2+)] that may impact subsequent synaptic efficacy. Using modified patch-clamp techniques to record from single neocortical nerve terminals, we report that physiologically relevant reductions of extracellular [Ca(2+)] ([Ca(2+)](o)) activate voltage-dependent outward currents. These outward currents are carried by a novel nonselective cation (NSC) channel that is indirectly inhibited by various extracellular agents (rank order potency, Gd(3+) > spermidine > Ca(2+) > Mg(2+), typical for [Ca(2+)](o) receptors). The identification of a Ca(2+) sensor-NSC channel pathway establishes the existence of a mechanism by which presynaptic terminals can detect and respond to reductions in cleft [Ca(2+)]. Activation of NSC channels by falls in [Ca(2+)](o) would be expected during periods of high activity in the neocortex and may modulate the excitability of the presynaptic terminal.     

Role of intracellular calcium channels of nerve terminals in the regulation of mediator secretion

The review is presented, analysing the modern state of knowledge about the role of intracellularly stored calcium of nerve terminals in regulation of quantal mediator secretion in synapses. The data are considered, concerning the properties of two Ca(2+)-channels superfamilies, i.e. the ryanodine receptors (RyR) and IP3-receptors, which are incorporated into the membrane of endoplasmic reticulum fragments. The localization of cisternae, containing RyR and IP3-receptors in neurons and nerve terminals are described. The data, demonstrating the pattern of calcium signalization in neurons and terminals after their interaction with specific blockers or activators of RyRs or IP3-receptors are presented. The facts, demonstrating that calcium induced calcium release via RyRs or IP3-receptors takes part in controlling spontaneous secretion of different types of vesicles in synaptic terminals and supports the slow and fast types of regulated exocytosis of synaptic vesicles, in the course of single or repetitive activity of central or peripheral synapses are analysed.     

Loading Drosophila nerve terminals with calcium indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca(2+)-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca(2+) changes in nerve terminals. However, these indicators have limited sensitivity to Ca(2+) and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca(2+) changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca(2+) indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca(2+) to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca(2+)-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca(2+) dynamics. Dextran-conjugation helps prevent Ca(2+) indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.     

Three-dimensional solution structure of omega-conotoxin TxVII, an L-type calcium channel blocker

We determined the three-dimensional structure of omega-conotoxin TxVII, a 26-residue peptide that is an L-type calcium channel blocker, by (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 411 distance constraints obtained from nuclear Overhauser effect connectivities, 20 torsion angle constraints, and 21 constraints associated with hydrogen bonds and disulfide bonds. The root-mean-square deviations about the averaged coordinates of the backbone atoms (N, C(alpha), C, and O) and all heavy atoms were 0.50 +/- 0.09 A and 0.99 +/- 0.13 A, respectively. The structure of omega-conotoxin TxVII is composed of a triple-stranded antiparallel beta-sheet and four turns. The three disulfide bonds in omega-conotoxin TxVII form the classical cystine knot motif of toxic or inhibitory polypeptides. The overall folding of omega-conotoxin TxVII is similar to those of the N-type calcium channel blockers, omega-conotoxin GVIA and MVIIA, despite the low amino acid sequence homology among them. omega-Conotoxin TxVII exposes many hydrophobic residues to a certain surface area. In contrast, omega-conotoxin GVIA and MVIIA expose basic residues in the same way as omega-conotoxin TxVII. The channel binding site of omega-conotoxin TxVII is different from those of omega-conotoxin GVIA and MVIIA, although the overall folding of these three peptides is similar. The gathered hydrophobic residues of omega-conotoxin TxVII probably interact with the hydrophobic cluster of the alpha(1) subunit of the L-type calcium channel, which consists of 13 residues located in segments 5 and 6 in domain III and in segment 6 in domain IV.     

The antagonism between botulinum toxin and calcium in motor nerve terminals

The effects of tetraethylammonium and manganese, which modify calcium entry into motor nerve terminals, have been studied during advanced stages of botulinum paralysis. Evidence has been obtained that the voltage-activated calcium current in the nerve endings is not significantly reduced by botulinum toxin. The depression of transmitter release that the toxin produces must arise at a later stage, at an intracellular site of the release mechanism.     

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.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca²⁺-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca²⁺ entry with Ca²⁺ ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca²⁺ influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca²⁺ channel activity and subsequent reduces Ca²⁺ entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity.     

LH-RH antagonist inhibits gonadal steroid secretion in vitro

This investigation was aimed at studying the direct action of LH-RH derivatives on gonadal function. Modulation by LH-RH antagonist (Ac-[D-beta-Nal1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]-LH-RH) and agonist of LH-RH (D-Ser(TBU)6, AzaGly10-LH-RH) and native LH-RH of HCG-stimulated steroidogenesis in testicular Leydig cells and luteal cells was studied in vitro. The LH-RH antagonist (3.2 X 10(-8) M) was found to change ED50 of HCG from 2 X 10(-11) M to 5.5 X 10(-11) M in the Leydig cell culture system. In addition, the antagonist was noted to override the stimulatory action of native LH-RH in Leydig cells. Furthermore, the agonist was found to augment HCG-provoked testosterone secretion. Similarly, the LH-RH antagonist at 10(-9) M blunted HCG-stimulated progesterone secretion in the luteal cell culture system and increased the ED50 of HCG from 8.7 X 10(-13) to 7.7 X 10(-12) M. In contrast to the Leydig cell culture system native LH-RH (10(-8) M) and the agonist (10(-8) M) increased the ED50 of HCG in luteal cells from 8.7 X 10(-13) M to 3 X 10(-12) M and 2.3 X 10(-12) M, respectively. Present data combine to suggest that LH-RH antagonists act at least partially at the gonadal level and may be clinically useful to inhibit Leydig cell and ovarian function.     

Inhibition of Escherichia coli chemotaxis by omega-conotoxin, a calcium ion channel blocker.

Escherichia coli chemotaxis was inhibited by omega-conotoxin, a calcium ion channel blocker. With Tris-EDTA-permeabilized cells, nanomolar levels of omega-conotoxin inhibited chemotaxis without loss of motility. Cells treated with omega-conotoxin swam with a smooth bias, i.e., tumbling was inhibited.     

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons

The effect of varying the external Mg²⁺ concentration on Ca²⁺ currents through voltage-operated Ca²⁺ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab,Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg²⁺ concentration on K⁺-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca²⁺ levels the high-voltage-activated Ca²⁺ currents were attenuated with increasing Mg²⁺ concentration, with 50% inhibition at 75 mM. Mg²⁺ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg²⁺ inhibition of the Ca²⁺ current was 55% at –10 mV and 30% at +20 mV. Secretion of CHH varied almost linearly with the log of Mg²⁺ concentration; in 2.4 mM Mg²⁺ it was double that in 24 mM Mg²⁺ and almost completely inhibited in 100 mM. Thus, Mg²⁺ produces a parallel inhibition of Ca²⁺ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.     

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals

Cytosolic Ca²⁺ buffers bind to a large fraction of Ca²⁺ as it enters a cell, shaping Ca²⁺ signals both spatially and temporally. In this way, cytosolic Ca²⁺ buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca²⁺ entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca²⁺ buffers in controlling pituitary Ca²⁺ signaling is poorly understood. Here, cytosolic Ca²⁺ buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca²⁺ indicator fluo-8 revealed stepwise increases in free Ca²⁺ after a series of brief depolarizing pulses in rapid succession. These Ca²⁺ increments grew larger as free Ca²⁺ rose to saturate the cytosolic buffers and reduce the availability of Ca²⁺ binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a K_d of 1.5–4.7 µM, and approximately half contained an additional higher-affinity buffer with a K_d of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca²⁺ buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca²⁺ signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.     

Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to omega-conotoxin and relatively insensitive to dihydropyridines. Optical studies with and without voltage-sensitive dyes

Extrinsic absorption changes exhibited by potentiometric dyes have established the ionic basis of the action potential in synchronously activated populations of nerve terminals in the intact neurohypophyses of amphibia and mammals (Salzberg et al., 1983; Obaid et al., 1983, 1985b). Also, large and rapid changes in light scattering, measured as transparency, have been shown to follow membrane depolarization and to be intimately associated with the release of neuropeptides from the nerve terminals of the mouse neurohypophysis (Salzberg et al., 1985; Gainer et al., 1986). We report some experiments that help to define the pharmacological profile of the calcium channels present in intact neurosecretory terminals of vertebrates. For these, we used the peptide toxin omega-conotoxin GVIA (1-5 microM) and the dihydropyridine compounds Bay-K 8644 and nifedipine (2-5 microM), together with the after-hyperpolarization of the nerve terminal action potential. This undershoot depends upon the activation of a calcium-mediated potassium channel, as suggested by its sensitivity to [Ca++]o and charybdotoxin. omega-conotoxin GVIA substantially reduced the after-hyperpolarization in neurosecretory terminals of Xenopus, while neither of the dihydropyridine compounds had any effect under conditions that mimic natural stimulation. The effects of these calcium channel modifiers on the action potential recorded optically from the terminals of the Xenopus neurohypophysis were faithfully reflected in the behavior of the light-scattering changes observed in the neurohypophysis of the CD-1 mouse. omega-conotoxin GVIA (5 microM) reduced the size of the intrinsic optical signal associated with secretion by 50%, while the dihydropyridines had little effect. These observations suggest that the type of calcium channel that dominates the secretory behavior of intact vertebrate nerve terminals is at least partially blocked by omega-conotoxin GVIA and is insensitive, under normal conditions, to dihydropyridines.