Synthesis and characterization of μ‐conotoxin IIIa

Abstract

p-Conotoxin 111 a, a voltage-dependent sodium channel neurotoxin, has been synthesised using solid-phase peptide synthesis employing 9-fluorenylmethoxycarbonyl chemistry. After cleavage from the resin, the peptide was isolated by reverse-phase HPLC and then the six acetamidomethyl groups were removed by treatment with mercuric acetate. The reduced product so formed was purified by reverse-phase HPLC. Protocols were developed to optimize the oxidation of the cysteine residues to form disulphide bonds. Protocols employed using air oxidation together with 2-mercaptoethanol were the most effective. As complete oxidation was never obtained the oxidised peptide was purified by reverse-phase HPLC. The acitivity of our products was monitored using [3H]saxitoxin binding to eel membranes. The oxidised product was able to completely block [3H]saxitoxin binding in a competitive manner. Lineweaver-Burke analysis of ['H]saxitoxin binding gave a K, of 1.5 nM, 1Cs0 was determined as 26.6 nM. It was also shown that the pure synthetic p-conotoxin IIIa had the same retention time on reversephase HPLC as the natural conotoxin IIIa. Thus an active toxin has been synthesised that can be used to probe sodium channels.Neurotoxins from various natural sources have been essential tools for labelling and physiologically manipulating the sodium channel [l]. Current understanding of toxin binding to the sodium channel implies the existence of five or more discrete toxin-binding sites. These binding sites are (a) the tetrodotoxin/saxitoxin-binding site, at which ligands inhibit ion transport, (b) the alkaloid-binding site at which toxins like batrachotoxin and veratridine bind to produce persistent activation of the sodium channel, (c) the North African scorpion a-toxinlsea anemone toxin-binding site where these reagents act to remove inactivation, (d) the American scorpion j-toxin site at which toxins mediate a shift in inactivation and (e) the ciguatoxin/ptychodiscus sitc; reagents binding here cause repetitive firing and persistent activation [2, 31.More recently a series of sodium-channel-specific toxins derived from the marine gastropod family Conidne have been described [4]. These purified polypeptide toxins from Conus geographus, the so-called p-conotoxins (also called geographutoxins) have a common structure and elicit a blockade of sodium currents by high-affinity binding to the tetrodotoxin/saxitoxin-binding site [5,6]. These toxins can block electric eel Electrophorus electricus and skeletal muscle sodium channels but not channels from brain, heart or nerve tissues [5, 7. 81. p-Conotoxins can be used to electrophysiologically distinguish between the different sodium channels that are observed during muscle development in a manner to the more widely described tetrodotoxin sensitivity [9, lo]. The amino acid sequences have been described and reveal that these toxins contain the rare amino acids hydroxyproline together with many cysteine residues [5, 61. The latter observation suggests that these toxins have a multi-looped structu...

Action of derivatives of .mu.-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates

“…All experiments using "native" toxin used synthetic peptide having the sequence of µ-conotoxin GUIA and previously shown to be indistinguishible in properties from the native toxin isolated by purification (Becker et al, 1989).…”

“…The raw peptides were oxidized and purified as recently described (Becker et al, 1989(Becker et al, ,1990). The molecular weight of each oxidized, purified analogue was checked by FABMS. For each analogue, the measured molecular weight corresponded to the predicted value within ±3 units, consistent with the correctness of the sequence and the complete removal of all side-chain protection groups.…”

α1‐Adrenergic stimulation causes Mg2+ release from perfused rat liver

“…Mg2+-induced changes in lipid order and conformation of renal (Na +,K÷)-ATPase have been described [71. O~l-Adrenergic stimulation of ouabain-sensitive K + uptake by perfused rat livers has been reported earlier [8], leaving open the possibility that release of membraneassociated Mg 2+ affects the regulation of (Na ÷ ,K÷)-ATPase.…”

Discovery of a New Analgesic Peptide, Leptucin, from the Iranian Scorpion, Hemiscorpius lepturus

“…In all deprotection methods, from the earliest established by Veber on cysteine-rich peptides [ 56 ], and then the modified Veber et al’s protocol (Becker et al’s protocol) [ 57 ], to the method of Eisapoor et al, which was the best method of deprotection [ 43 ], it’s a common point. In these methods, the removal of the ACM group with mercury acetate had been performed in pH 4 [ 43 , 56 , 57 ].…”