Voltage-gated sodium channels (Na_Vs), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na_V ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na_V subtypes (rat Na_V1.4 and human Na_V1.5). Two of these compounds, BTX-B and BTX-^cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na_V modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na_Vs (BacNa_Vs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na_V gating mechanisms and designing allosteric regulators of Na_V activity.

Voltage-gated sodium channels (Na_Vs), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na_V ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na_V subtypes (rat Na_V1.4 and human Na_V1.5). Two of these compounds, BTX-B and BTX-^cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na_V modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na_Vs (BacNa_Vs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na_V gating mechanisms and designing allosteric regulators of Na_V activity.