Multiple, Distributed Interactions of μ-Conotoxin PIIIA Associated with Broad Targeting among Voltage-Gated Sodium Channels

Abstract

The first μ-conotoxin studied, μCTX GIIIA, preferentially blocked voltage-gated skeletal muscle sodium channels, Na(v)1.4, while μCTX PIIIA was the first to show significant blocking action against neuronal voltage-gated sodium channels. PIIIA shares >60% sequence identity with the well-studied GIIIA, and both toxins preferentially block the skeletal muscle sodium channel isoform. Two important features of blocking by wild-type GIIIA are the toxin's high binding affinity and the completeness of block of a single channel by a bound toxin molecule. With GIIIA, neutral replacement of the critical residue, Arg-13, allows a residual single-channel current (~30% of the unblocked, unitary amplitude) when the mutant toxin is bound to the channel and reduces the binding affinity of the toxin for Na(v)1.4 (~100-fold) [Becker, S., et al. (1992) Biochemistry 31, 8229-8238]. The homologous residue in PIIIA, Arg-14, is also essential for completeness of block but less important in the toxin's binding affinity (~55% residual current and ~11-fold decrease in affinity when substituted with alanine or glutamine). The weakened dominance of this key arginine in PIIIA is also seen in the fact that there is not just one (R13 in GIIIA) but three basic residues (R12, R14, and K17) for which individual neutral replacement enables a substantial residual current through the bound channel. We suggest that, despite a high degree of sequence conservation between GIIIA and PIIIA, the weaker dependence of PIIIA's action on its key arginine and the presence of a nonconserved histidine near the C-terminus may contribute to the greater promiscuity of its interactions with different sodium channel isoforms.

Orientation of μ-Conotoxin PIIIA in a Sodium Channel Vestibule, Based on Voltage Dependence of Its Binding

“…The basic residues of PIIIA (McArthur et al, 2011), and of other CTXs, play a vital role in toxin binding to the pore of voltage-gated sodium channels and in blocking current through the channel. Given the high sequence identity between GIIIA and PIIIA, most of the important basic residues are conserved (Fig.…”

Interactions of Key Charged Residues Contributing to Selective Block of Neuronal Sodium Channels by μ-Conotoxin KIIIA

“…Currents were elicited with a 2-s prepulse to Ϫ140 mV to remove inactivation followed by a test pulse to Ϫ10 mV for 10 ms repeated every 5 s during toxin application and washout to record kinetics. Single-channel bilayer experiments using batrachotoxinmodified skeletal muscle Na v channel at steady state were carried out as described previously (McArthur et al, 2011a).…”

“…This residual single-channel current may be permitted by the absence of arginine and lysine residues in its N-terminal segment (residues 1-6), given that the PIIIA R12A derivative shows a small residual current similar to that for KIIIA (McArthur et al, 2011a). Even though KIIIA binds to the sodium-channel pore site 1, either tetrodotoxin or saxitoxin can bind simultaneously ), increasing the range of possible pharmacological actions of KIIIA by its use in combination with the smaller pore blockers.…”

“…Channel mutants were selected based on sequence comparisons (Fig. 1B) and previous docking simulations of CTXs GIIIA and PIIIA (Choudhary et al, 2007;McArthur et al, 2011a). We focused on two positions: 1) the outer ring charge in domain III, which is absent in Na v 1.7 but present in Na v 1.4 and Na v 1.2; and 2) the aromatic residue in domain I, adjacent to the DEKA locus, which has important implications for tetrodotoxin and saxitoxin block (Santarelli et al, 2007) and thus could be important in KIIIA block.…”

Large-scale Transcriptomic and Proteomic Data Mining of Conopeptides and Cysteine-rich Venom Toxins

“…174 With Arg 14 positioned in the permeation pathway, PIIIA causes steric and/or electrostatic occlusion of the channel pore. 175 Replacement of His 19 by Gln increases the selectivity of PIIIA for Na V 1.4 over Na V 1.2, 175 suggesting that His 19 , which is highly conserved among m-conotoxins, increases selectivity for the neuronal subtype. Interestingly, a recent study demonstrated that PIIIA isoforms with different cysteine connectivity (i.e., without changes in the primary structure) can be more potent blockers of Na V 1.4.…”