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.