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.

Effects of stimulus intensity on the inhibition by omega-conotoxin GVIA and neomycin of K(+_-evoked [3H]norepinephrine release from hippocampal brain slices and synaptosomal calcium influx.

The effects of various K+ concentrations on the inhibition of [3H]norepinephrine release from rat hippocampal brain slices and evoked synaptosomal 45Ca2+ influx by omega-conotoxin GVIA (omega-CgTx) and neomycin were examined. K+ (15-75 mM) caused a concentration-dependent release of [3H]norepinephrine that was greater than 90% dependent on extracellular calcium. The ability of omega-CgTx to inhibit [3H]norepinephrine release was optimal at 25 mM K+ and was reduced substantially at higher concentrations of K+. omega-CgTx maximally inhibited [3H]norepinephrine release by 49% (15 mM K+), 58% (25 mM K+), 22% (50 mM K+), and 12% (75 mM K+). In contrast, neomycin caused a concentration-dependent and virtually complete inhibition of [3H]norepinephrine release at all concentrations of K+, with IC50 values of 210 microM (15 mM K+), 150 microM (25 mM K+), 450 microM (50 mM K+), and 1500 microM (75 mM K+). omega-CgTx (1 microM) had little effect (10% or less inhibition) on hippocampal synaptosomal 45Ca2+ influx at any concentration of K+, whereas 3 mM neomycin caused at least 75% inhibition of 45Ca2+ influx, with the largest inhibition (96%) occurring at 25 mM K+. The results suggest that increasing stimulus intensity decreases the contribution of N-type voltage-sensitive calcium channels (VSCC) in mediating K(+)-evoked release of [3H]norepinephrine. The comparative absence of omega-CgTx-sensitive synaptosomal 45Ca(2+)-influx sites suggests that N-type calcium channels are a small subset of channels in rat hippocampal synaptosomes. The demonstration that neomycin can inhibit omega-CgTx-sensitive and -insensitive neurotransmitter release and calcium influx suggests that neomycin may block N-type VSCC as well as non-N-type VSCC.

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses.

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.