Differential effects of modified batrachotoxins on voltage-gated sodium channel fast and slow inactivation

Voltage-gated sodium channels (Na_Vs) 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. Herein, we detail studies with batrachotoxin (BTX), a potent steroidal amine, and three ester derivatives prepared through de novo synthesis against recombinant Na_V subtypes (rNa_V1.4 and hNa_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-n-heptynoate ester-is a conspicuous outlier, eliminating fast but not slow inactivation. This property differentiates BTX-yne among other Na_V modulators as a unique reagent 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.