Background and Purpose: Voltage‐gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore‐bearing α‐subunit (Na_V1) and one or two accessory β‐subunits (Na_Vβs). Neurons in mammals can express one or more of seven isoforms of Na_V1 and one or more of four isoforms of Na_Vβ. The peptide μ‐conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in Na_V1. Hitherto, the effects of Na_Vβ‐subunit co‐expression on the activity of these toxins have not been comprehensively assessed.Experimental Approach: Four μ‐conotoxins (μ‐TIIIA, μ‐PIIIA, μ‐SmIIIA and μ‐KIIIA), TTX and STX were tested against Na_V1.1, 1.2, 1.6 or 1.7, each co‐expressed in Xenopus laevis oocytes with one of Na_Vβ1, β2, β3 or β4 and, for Na_V1.7, binary combinations of thereof.Key Results: Co‐expression of Na_Vβ‐subunits modifies the block by μ‐conotoxins: in general, Na_Vβ1 or β3 co‐expression tended to increase k_on (in the most extreme instance by ninefold), whereas Na_Vβ2 or β4 co‐expression decreased k_on (in the most extreme instance by 240‐fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by Na_Vβ‐subunit co‐expression. Tests of Na_Vβ1 : β2 chimeras co‐expressed with Na_V1.7 suggest that the extracellular portion of the Na_Vβ subunit is largely responsible for altering μ‐conotoxin kinetics.Conclusions and Implications: These results are the first indication that Na_Vβ subunit co‐expression can markedly influence μ‐conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. μ‐Conotoxins could, in principle, be used to pharmacologically probe the Na_Vβ subunit composition of endogenously expressed VGSCs.

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of ne...

Voltage-gated sodium channels (VGSCs) consist of a pore-forming α-subunit and regulatory β-subunits. Several families of neuroactive peptides of Conus snails target VGSCs, including μO-conotoxins and μ-conotoxins. Unlike μ-conotoxins and the guanidinium alkaloid saxitoxin (STX), which are pore blockers, μO-conotoxins MrVIA and MrVIB inhibit VGSCs by modifying channel gating. μO-MrVIA/B can block Na_V1.8 (a tetrodotoxin-resistant isoform of VGSCs) and have analgesic properties. The effect of Na_Vβ-subunit coexpression on susceptibility to block by μO-MrVIA/B and STX has, until now, not been reported. Here, we show that β1-, β2-, β3-, and β4-subunits, when individually coexpressed with Na_V1.8 in Xenopus laevis oocytes, increased the k_on of the block produced by μO-MrVIB (by 3-, 32-, 2-, and 7-fold, respectively) and modestly decreased the apparent k_off. Strong depolarizing prepulses markedly accelerated MrVIB washout with rates dependent on β-subunit coexpression. Thus, coexpression of β-subunits with Na_V1.8 can strongly influence the affinity of the conopeptide for the channel. This observation is of particular interest because β-subunit expression can be dynamic, e.g., β2-expression is up-regulated after nerve injury (J Neurosci, 25:10970–10980, 2005); therefore, the effectiveness of a μO-conotoxin as a channel blocker could be enhanced by the conditions that may call for its use therapeutically. In contrast to MrVIB9s action, the STX-induced block of Na_V1.8 was only marginally, if at all, affected by coexpression of any of the β-subunits. Our results raise the possibility that μO-conotoxins and perhaps other gating modifiers may provide a means to functionally assess the β-subunit composition of VGSC complexes in neurons.

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 μ-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 μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-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 μ-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.

Significance: A biochemically unique cone snail venom peptide has been characterized that may be used to probe unexplored but important features of the diverse voltage-gated Na channel isoforms that underlie electrical signaling in the nervous system. This peptide has a unique posttranslational modification (S-cysteinylated cysteine) and blocks sodium channels by forming a disulfide bond with the channel at a distinctive binding site. Because block by the peptide is prevented when specific β-subunits are coexpressed, this neurotoxin has potential for assessing which β-subunits are present in native Na channels. Peptide activity depends on the oxidation state of extracellular Cys residues on the channel. Thus, this peptide can also be used to monitor the oxidation state of the targeted Na channels.