Voltage-dependent inhibition of N- and P-type calcium channels by the peptide toxin omega-grammotoxin-SIA.

We studied the mechanism by which the peptide omega-grammotoxin-SIA inhibits voltage-dependent calcium channels. Grammotoxin at concentrations of > 50 nM completely inhibited inward current carried by 2 mM barium through P-type channels in rat cerebellar Purkinje neurons when current was elicited by depolarizations up to +40 mV. However, outward current (carried by internal cesium) elicited by depolarizations to > +100 mV was either unaffected or enhanced in the presence of toxin. Tail current activation curves showed that grammotoxin shifted the steady state voltage dependence of channel activation by approximately +40 mV. Activation in the presence of toxin was far slower in addition to having altered voltage dependence. Grammotoxin also inhibited N-type calcium channels in rat and frog sympathetic neurons, with changes in channel voltage dependence and kinetics nearly identical to those of P-type channels. Experiments with monovalent ions as the only charge carriers showed that toxin effects on channel activation and kinetics depended on voltage, not on direction of current flow or on the current-carrying ion. Repeated trains of large depolarizations relieved toxin inhibition, as if toxin affinity for activated channels were low. The effects of grammotoxin on gating of P-type channels are very similar to those of omega-Aga-IVA, but combined application of the two toxins showed that grammotoxin binding is not prevented by saturating binding of omega-Aga-IVA. We conclude that grammotoxin potently inhibits both P-type and N-type channels by impeding channel gating and that grammotoxin binds to distinct or additional sites on P-type channels compared with omega-Aga-IVA.

Complete and reversible block by omega-grammotoxin SIA of glutamatergic synaptic transmission between cultured rat hippocampal neurons.

omega-Grammotoxin SIA (omega-GsTx SIA), a peptide isolated from tarantula venom, inhibits synaptosomal Ca2+ influx and neurotransmitter release, and blocks N-, P-, and Q-type voltage-gated Ca2+ channels. The whole-cell patch-clamp was used to record glutamatergic excitatory post-synaptic currents (EPSCs) evoked by extracellular stimulation of presynaptic neurons in primary rat hippocampal cultures. EPSCs displayed rapid kinetics and were blocked by CNQX. omega-Conotoxin (1 microM) GVIA inhibited EPSCs by 46%, while 30 nM and 1 microM omega-agatoxin IVA produced 12% and 69% inhibition, respectively, consistent with coupling of N-, P- and Q-type Ca2+ channels to glutamatergic synaptic transmission. omega-GsTx SIA (1 microM) rapidly, completely, and reversibly blocked glutamatergic EPSCs, but did not affect currents evoked by bath application of kainate. Thus, omega-GsTx SIA blocks glutamatergic synaptic transmission by blocking presynaptic voltage-gated Ca2+ channels. omega-GsTx SIA is the only agent that blocks selectively and reversibly the Ca2+ channels coupled to glutamate release. omega-GsTx SIA provides a unique and powerful tool for experiments requiring recovery of function following presynaptic block of synaptic transmission.

Comparative actions of synthetic omega-grammotoxin SIA and synthetic omega-Aga-IVA on neuronal calcium entry and evoked release of neurotransmitters in vitro and in vivo.

The effects of synthetic omega-grammotoxin SIA (omega-GsTxSIA) and synthetic omega-Aga-IVA were tested in in vitro and in vivo neurochemical assays that are reflective of voltage-sensitive calcium channel function. Synthetic omega-GsTx SIA inhibited K(+)-evoked rat and chick synaptosomal 45Ca2+ flux, K(+)-evoked release of [3H]D-aspartate and [3H]norepinephrine from rat hippocampal brain slices and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices with potency values that were comparable to those found previously with omega-GsTx SIA purified from the venom of the tarantula spider Grammostola spatulata. These results indicate that trace contaminants do not account for the pharmacology of purified omega-GsTx SIA. omega-GsTx SIA caused a complete inhibition of rat synaptosomal 45Ca2+ flux and hippocampal slice [3H]D-aspartate release, whereas omega-Aga-IVA caused a maximal inhibition of approx 75%. omega-GsTx SIA and omega-Aga-IVA caused an identical partial inhibition of K(+)-evoked increases of intracellular calcium in cortical neurons in primary culture. The addition of nitrendipine to either omega-GsTx SIA or omega-Aga-IVA resulted in an additive and virtually complete inhibition of the cortical neuron intracellular calcium response. In in vivo microdialysis studies, the K(+)-evoked release of glutamate from hippocampus of awake freely moving rats was inhibited with the following rank order of potency: omega-conotoxin GVIA > omega-GsTx SIA > omega-Aga-IVA. Complete inhibition of K(+)-evoked hippocampal glutamate release was observed with 300 nM omega-conotoxin GVIA and 3 microM omega-GsTx SIA. In urethane anesthetized rats, omega-CgTx GVIA caused a partial inhibition, whereas omega-GsTx SIA caused a concentration-dependent and complete inhibition, of basal serotonin release in the hippocampus. Therefore, omega-GsTx SIA was shown to inhibit responses that are sensitive to omega-conotoxin GVIA, omega-Aga-IVA and omega-conotoxin MVIIC, consistent with the notion that omega-GsTx SIA inhibits N-, P- and Q-type high threshold voltage-sensitive calcium channels.

Omega-grammotoxin SIA blocks multiple, voltage-gated, Ca2+ channel subtypes in cultured rat hippocampal neurons.

Omega-Grammotoxin SIA is a peptide isolated from tarantula venom on the basis of its ability to block the voltage-gated Ca2+ channels that mediate glutamate release. To determine the Ca2+ channel subtype selectivity of omega-grammotoxin SIA, whole-cell Ba2+ current (IBa) was measured in cultured rat hippocampal neurons. Selective Ca2+ channel blockers were used to identify components of IBa mediated by Ca2+ channel subtypes. omega-Agatoxin IVA at 30 nM, 1 microM omega-conotoxin GVIA, and 3 microM omega-contoxin MVIIC, applied consecutively, each elicited a fractional increase in the cumulative block of IBa, identifying components of IBa mediated by P-, N-, and Q-type calcium channels. omega-Grammotoxin at 1 microM, a maximally effective concentration, blocked 52% of IBa. omega-Conotoxin MVIIC and the combination of omega-conotoxin GVIA and micromolar omega-agatoxin IVA blocked 52% and 54% of IBa, respectively, and block of IBa by omega-grammotoxin SIA was mutually occlusive of block of IBa by either treatment, both of which block N-, P-, and Q-type Ca2+ channels. The L channel blocker nimodipine produced identical block of IBa in the presence and absence of omega-grammotoxin SIA. These results indicate that omega-grammotoxin SIA blocks N-, P-, and Q-type but not L-type voltage-gated calcium channels. Block of IBa by omega-grammotoxin SIA was faster in onset and less sensitive to external divalent cation concentrations than was block by omega-conotoxin MVIIC, and it was rapidly and substantially reversible. Rapid onset, relative insensitivity to divalent cation concentrations, and reversibility render omega-grammotoxin SIA a useful tool for inhibition of neuronal voltage-gated Ca2+ channels.

Characterization of presynaptic calcium channels with omega-conotoxin MVIIC and omega-grammotoxin SIA: role for a resistant calcium channel type in neurosecretion.

The peptide Ca2+ channel antagonists omega-conotoxin (omega-CTX) MVIIC and omega-grammotoxin (omega-GTX) SIA were studied by measuring their effects on the release of [3H]glutamate from rat brain synaptosomes. The pseudo-first-order association constant for omega-CTX MVIIC (1.1 x 10(4) M-1 sec-1) was small, relative to that for omega-GTX SIA (3.6 x 10(5) M-1 sec-1). Equilibrium experiments showed that omega-CTX MVIIC blocked approximately 70% of Ca(2+)-dependent glutamate release evoked by 30 mM KCl (IC50 approximately 200 nM), whereas omega-GTX SIA virtually eliminated release, with lower potency (IC50 approximately 700 nM). At stronger depolarizations (60 mM KCl), neither toxin (at 1 microM) showed significant block of release, but when these or other Ca2+ channel antagonists (omega-CTX GVIA or omega-agatoxin IVA) were used in combination a substantial fraction of release was blocked. [3H]Glutamate release that was resistant to omega-CTX MVIIC was characterized with respect to its sensitivity to block by omega-GTX SIA and the inorganic blocker Ni2+. Both omega-GTX SIA and Ni2+ were relatively weak blockers of the resistant release. These results suggest that a previously uncharacterized Ca2+ channel exists in nerve terminals and can be distinguished on the basis of its resistance to omega-CTX MVIIC and its weak sensitivity to omega-GTX SIA and Ni2+. Thus, at least three channel types (P, N, and a "resistant" type) contribute to excitation-secretion coupling in nerve terminals.

omega-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons.

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca2+]i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca2+]i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. omega-Conotoxin GVIA (omega-CgTx, 100 nM) inhibited action-potential-mediated Ca2+ influx by 79%, while nitrendipine (1 microM) had little effect. omega-Grammotoxin SIA (omega-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca2+ influx as effectively as did omega-CgTx, suggesting that omega-GsTx blocks N-type Ca2+ channels. In contrast to block by omega-CgTx, the block produced by omega-GsTx reversed upon washout of the peptide. omega-GsTx (270 nM) blocked 80%, and omega-CgTx (1 microM) blocked 64%, of whole-cell Ca2+ current (ICa) elicited by step depolarization to 0 mV from a holding potential of -80 mV. omega-GsTx completely occluded inhibition of ICa by omega-CgTx. However, when applied after omega-CgTx, omega-GsTx produced an additional inhibition of 27%, indicating that omega-GsTx also blocked a non-N-type Ca2+ channel. BayK8644 (1 microM) elicited an increase in ICa in the presence of maximally effective concentrations of omega-GsTx, suggesting that omega-GsTx does not block L-type channels. Thus, omega-GsTx displays a selectivity for Ca2+ channel subtypes which should prove useful for studying Ca2+ channels and Ca(2+)-channel-mediated processes.

The influence of amino acid sequence on the fibrillogenicity and amyloidogenicity of the carboxy-terminus of beta-amyloid precursor protein.

C-APP, a synthetic peptide corresponding to the C-terminal 20 amino acids of beta-amyloid precursor protein, forms amyloid fibrils in vitro. We investigated the effect of altering the C-APP sequence or deleting part of it on its ability to form amyloid fibrils. Substituting any single amino acid in the C-APP sequence with alanine did not prevent the formation of CAPP-like fibrils. Peptides with single or multiple substitutions that included T11, F14, F15, or Q19 showed reduced fibril-forming capacity while those with K1 and/or K13 replaced with alanine or glutamic acid showed enhanced capacity. When P10 or F14 was replaced with alanine, the fibrils were less congophilic than C-APP fibrils. All of the truncated peptides that were able to form fibrils contained at least 9 amino acids from the N-terminus of C-APP or amino acids 7-20 from the C-terminus. However, several peptides that met these criteria, but started at Q3 or contained only 2-4 amino acids C-terminal to P-10, failed to form many or typical fibrils. Peptides that contained the C-APP sequence plus 5-20 adjacent amino acids from the beta-amyloid precursor protein formed fibrils less readily than C-APP and most of the fibrils were not congophilic. The exception was CAPP-30, which formed moderate amounts of congophilic fibrils resembling C-APP fibrils morphologically. Therefore, proteolysis which releases C-APP from these peptides (except CAPP-30) would be predicted to enhance their amyloidogenicity. These results suggest that several features of C-APP peptide may be important in fibril formation. One of these features is the length of the peptide, with lengths of about 10, 20, or 30 amino acids, favoring fibril formation.

Isolation and pharmacological characterization of omega-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses.

omega-Grammotoxin SIA, a peptidergic blocker of voltage-sensitive calcium channel (VSCC) responses, was purified from Grammostola spatulata (tarantula spider) venom by reverse phase high performance liquid chromatography. Protease-sensitive biological activity was monitored by determining the inhibition of K(+)-stimulated influx of 45Ca2+ into rat brain synaptosomes. Electrospray mass spectrometry indicated an average molecular mass of 4109.2 Da for the native peptide. Chemical reduction of omega-grammotoxin SIA indicated the presence of three disulfide bridges. Primary sequence data confirmed the existence of six cysteine residues and 36 residues in total, with an average theoretical molecular mass of 4109.7 Da for the amidated carboxyl-terminal species. The biological profile of omega-grammotoxin SIA indicated virtually complete blockade of presynaptic vertebrate N-type as well as P-type VSCC responses. Specifically, omega-grammotoxin SIA caused a concentration-dependent and virtually complete inhibition of K(+)-evoked influx of 45Ca2+ into either rat or chick brain synaptosomes. Similar inhibition profiles were generated for the inhibition of release of either D-[3H]aspartate or [3H]norepinephrine from rat hippocampal or [3H]norepinephrine from chick cortical brain slice preparations evoked by K+ depolarization. As reported earlier, omega-grammotoxin SIA did not inhibit 125I-omega-conotoxin GVIA, [3H]PN 200-110, or [3H]desmethoxyverapamil binding to neuronal membrane fragments. To our knowledge, omega-grammotoxin SIA is the first ligand identified to block putative N-channel function without displacement of 125I-omega-conotoxin GVIA. omega-Grammotoxin SIA thus represents a novel vertebrate VSCC antagonist that inhibits neuronal N- and P-type VSCC responses.

Effects of site-specific acetylation on omega-conotoxin GVIA binding and function.

Chemical modification of omega-conotoxin GVIA (omega-CgTXGVIA) was performed using nonsaturating concentrations of acetic anhydride to generate seven distinct derivatives. Following separation of these peptides using reverse-phase HPLC (RP-HPLC), their individual molecular weights were determined using fast bombardment mass spectrometry (FAB-MS). Three peptides contained a single acetylated amino group, three possessed two acetylated amino groups, and the last contained three acetylations. For each peptide, the specific site of acetylation was confirmed using a scheme of tryptic digestion, under nonreducing conditions, followed by RP-HPLC and FAB-MS. Biological profiles for each peptide were obtained by analyzing their capacity to displace native 125I-omega-CgTx GVIA binding to rat hippocampal membranes and to block K(+)-stimulated 45Ca2+ influx into chick brain synaptosomes. The data indicate that successive additions of acetyl moieties to omega-CgTx GVIA lead to a loss of both binding affinity and Ca2+ influx inhibitory potency. Within the monoacetylated series, acetylation of the amino terminal of Cys-1, as compared to the epsilon-amino group of either Lys-2 or Lys-24, leads to the greatest shift in potency. In summary, these results indicate that basic (i.e., primary amino) groups, which are brought into close proximity as a result of disulfide bridging, are important in the functional blockade of neuronal Ca2+ channels by omega-CgTx GVIA.

Identification in rodents and other species of an mRNA homologous to the human beta-amyloid precursor.

The isolation and sequencing of the core peptide (beta-amyloid) found in the plaques of patients with Alzheimer's disease has allowed the identification of a cDNA for the precursor protein. Using a human cDNA clone for this beta-amyloid material, we have identified an homologous mRNA (3.8 kb) in brain tissue obtained from 8 additional species. We have also determined its distribution in 7 brain regions and 12 organs obtained from rodents. A prominent, second mRNA species (2.2 kb) has been identified in rat non-neuronal tissues. The beta-amyloid gene is amply expressed in the brain of all vertebrates tested and in most rodent organs, indicating that it encodes a highly conserved and ubiquitous protein.

Expression of rat brain excitatory amino acid receptors in Xenopus oocytes.

Xenopus laevis oocytes when injected with rat brain mRNA synthesize neuronal receptors that can be analyzed electrophysiologically. After a post-injection incubation period of 24-72 hours, L-glutamic acid, kainic acid and quisqualic acid caused a dose dependent (10-100 microM) depolarization of the oocyte membrane. The voltage and conductance changes associated with kainate activation were distinguishable from those seen for L-glutamate or quisqualate. There was no response to L-aspartate application and an inconsistent response to N-methyl-D-aspartate. Upon fractionation of the mRNA on sucrose gradients, transcripts greater than 2 Kb in length were obligatory for the synthesis of excitatory amino acid receptors. The electrophysiological response of injected oocytes exposed to L-glutamate was similar to that of native oocytes when exposed to muscarinic agents. This similarity may reflect the activation of the same ionophore and suggests that the active mRNA fraction for glutamate responsiveness either encodes for a binding protein that can be assembled along with native ion channels into the oocyte membrane or encodes for a glutamate binding site with a similar channel.

Effects of amphipathic drugs on L'[3H]glutamate binding to synaptic membranes and the purified binding protein.

Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding of L-[3H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibited L-[3H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca2+-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca2+ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na+-dependent L-glutamate transport in these membrane preparations. The importance of Ca2+ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.

Ethanol effects on synaptic glutamate receptors and on liposomal membrane structure.

Exposure of synaptic plasma membranes to 50 mM ethanol in vitro brought about a 3.5 degrees C decrease in the transition temperature of the high affinity glutamate binding process in these membranes. Ethanol had no effect on the energy of activation of glutamate binding below the transition temperature but decreased the energy of activation above the transition temperature. Electron paramagnetic resonance (EPR) studies of lipid organization of egg lecithin and bovine brain phospholipid liposomes indicated that ethanol at low concentrations (0.04--2 mM) caused small increases in the rigidity of the membrane near the surface. At higher concentrations (0.04--2 M) ethanol brought about increasing fluidization of both the surface and inner areas of the bilayer. Even at 4 mM concentration ethanol enhanced the ordered to fluid state transition of liposome membranes as shown by a 5.2 degrees C and 1.9 degrees C decrease in the transition temperatures of the membrane determined with the cholestane EPR probe.