Differential Effects of Modified Batrachotoxins on Voltage-gated Sodium Channel Fast and Slow Inactivation

Voltage-gated sodium channels (NaVs), 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 NaV 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 de novo 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 NaV subtypes (rat NaV1.4 and human NaV1.5). 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-n-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported NaV modulator that separates inactivation processes. These findings are supported by functional studies with bacterial NaVs (BacNaVs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding NaV gating mechanisms and designing allosteric regulators of NaV activity.