Abstract
Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).