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

Abstract

We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.

Structure-Activity Relationships of Hainantoxin-IV and Structure Determination of Active and Inactive Sodium Channel Blockers

“…Evidently the positively charged side chains are located on the surface of the peptide. In particular, the surface of loop IV (Arg 26 27 , and Arg 29 in HNTX-IV were the crucial residues for its blocking activity. Fig 7A, a shows the surface profile of native HNTX-IV and the presentation identifying the four interesting residues.…”

“…Many studies have revealed that the binding interface of the toxin molecule is rather wide, and multiple amino acid residues of toxins are involved in the interaction with Na ϩ channels (5, 6, 25). The NMR structure of HNTX-IV displayed that Ser 12 , Arg 26 , Lys 27 , and Arg 29 were clustered on one side of the molecule with their side chains being more surface-exposed ( Fig. 7A, a).…”

“…So the four spider toxins (HNTX-III-V and HWTX-IV) define a new class of spider toxins affecting VGSCs. The three-dimensional solution structure of HWTX-IV has been determined using two-dimensional 1 H NMR spectroscopy, and it was hypothesized that the positively charged residues of loop IV (residues 25-29), espe-cially Arg 26 , were crucial to its binding to the neuronal TTX-S VGSCs (8). However, this hypothesis has not been supported by the results of experiments with mutants.…”

“…1 H NMR analysis showed similar molecular conformations for native HNTX-IV and synthetic mutants. Pharmacological studies indicated that residues Ser 12 and Arg 26 were not important for the activities, while Lys 27 and Arg 29 were critical for the bioactivities. These results provide useful information for structure-activity relationships of toxins like HNTX-IV.…”

Conotoxins: from the biodiversity of gastropods to new drugs

“…Эти токсины избирательно блокируют натриевые каналы скелетных мышц (Nav1.4) и обладают существенно меньшим сродством к натриевым каналом других типов [52,53]. Структурно-функциональные исследования показали сложный характер взаимодействия GIIIA и GIIIB с порой натриевого канала, в котором участвуют несколько аминокислотных остатков конотоксина.…”