Batrachotoxin Sensitive Sodium Channels in Toxic Birds Challenge "Target Mutation" Strategy of Toxin Autoresistance

Voltage-gated sodium channels (NaVs) are essential for muscle and nerve activity, and are therefore prime targets of natural toxins. Batrachotoxin (BTX) is uniquely potent among NaV-directed alkaloids. Southern and Central American Phyllobates poison dart frogs and multiple species of New Guinean toxic birds accumulate dietary BTX without self-poisoning. Two broad autoresistance models have been proposed: 1) "target mutation," in which NaV substitutions reduce BTX action, and 2) "toxin sequestration," in which high-affinity binding proteins or compartmentalization limit toxin access to channels. A recent report identified two NaV1.4 substitutions, D1050N and S1568P, under positive selection in toxic birds, including two newly discovered BTX-bearing birds, Pachycephala schlegelii and Aleadryas rufinucha, and suggested a "target mutation" strategy for these passerines. Here, we test that hypothesis using structure-guided mapping and electrophysiology. The substitutions map to solvent-exposed positions tens of angstroms away from inner-cavity BTX sites, inconsistent with direct effects on BTX binding. Heterologously expressed mutant channels exhibit wild-type activation and steady-state inactivation, lacking the biophysical "costs" typical of pore-lining resistance mutations. Importantly, all constructs remain fully BTX-sensitive, showing canonical shifts in activation and persistent currents upon toxin exposure. These data argue that NaV1.4 substitutions do not confer BTX autoresistance in toxic birds. This finding, together with prior work in BTX-carrying frogs and birds, is consistent with a generalized sequestration model. Identifying the responsible BTX-binding factors could enable antidote design and broaden strategies for neutralizing NaV-targeting toxins.