The tetrodotoxin receptor of voltage-gated sodium channels--perspectives from interactions with micro-conotoxins

Abstract

Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide micro-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of micro-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain micro-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel's voltage sensor. Most recently, the relatively small micro-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.

Keywords: conopeptides; guanidinium toxins; pore block.

Figure 1

Steady-state recordings from a single, rat skeletal muscle sodium channel enable discrete recognition and analysis of blocking events induced by derivatives of both STX and the conotoxin, μCTX GIIIA. The channel has been modified by batrachotoxin to prevent inactivation. Voltage-dependent activation gating and block by site 1 ligands are retained. (a) Typical all-or-none block, resulting from guanidinium toxins, such as TTX and STX, exemplified by the saxitoxin derivative, decarbamoyl-STX (dcSTX); (b) After addition of peptide μCTX GIIIA[R13Q], prominent partial blocking events are seen, reducing current by ~70%. The fully blocked/closed level is indicated by the solid line; the partially blocked level produced by R13Q is indicated by the dotted line; (c) After increasing the concentrations of both dcSTX and R13Q, the channel is bound most of the time, but in a manner consistent with simple, competitive binding, each fully blocked state (dcSTX-bound), and each partially blocked state (R13Q-bound), is preceded by an unblocked state, which is relatively brief at these high concentrations of the toxins. Adapted from French et al., Neuron, 1996 [18].

Figure 2

μCTX GIIIA, with its nominal net charge of +6, shifts voltage dependent gating charge movement to the right, which is consistent with the cationic peptide impeding outward movement of the positive charge on the channel’s voltage sensor [21]. This is consistent with shifts in activation of both single channels in lipid bilayers and whole-cell currents [18]. Recording from fused tsA-201 cells expressing rat skeletal muscle VGSCs (rNa_V1.4) [22].

Figure 3

Reaction scheme showing simultaneous binding of TTX and μCTX KIIIA. Reproduced by permission from Zhang et al. 2009, Channels [25].