Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on "toxin sponge" proteins.

Nathan D. Rossen,J. Du Bois,Catherine E. Garrison,John P. Dumbacher,Megan E. Kobiela,Daniel L. Minor,Zhou Chen,Fayal Abderemane-Ali,Lauren A. O’Connell,Claire M. Colleran,Robert A. Craig

doi:10.1085/jgp.202112872

Abstract

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX "toxin sponge" protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

Highlights

Many organisms harbor various small-molecule toxins that target ion channels as a means of defense from predation (Savitzky et al, 2012)
We found that the DIVS6 N→T variant is absent from Pitohui and poison frog NaVs, incurs a strong cost that compromises channel function, and fails to produce BTX-resistant channels when tested in the context of poison frog NaVs
Whole-cell patch-clamp electrophysiology of Pitohui uropygialis meridionalis (Pum) NaV1.4 and Pum NaV1.5 transfected into HEK293 cells demonstrated that both have fast voltage-dependent activation followed by a fast and complete voltage-dependent inactivation typical of NaVs (Fig. 1, a and b; Fig. S3, a and b; Table 1; and Table S1), similar to Homo sapiens (Hs) NaV1.4 recorded under identical conditions (Fig. 1, c and d; Table 1; and Table S1)

Summary

Introduction

Many organisms harbor various small-molecule toxins that target ion channels as a means of defense from predation (Savitzky et al, 2012). Batrachotoxin (BTX), a dietacquired (Daly et al, 1994b; Daly et al, 1994a; Dumbacher et al, 2004) steroidal amine found in distantly related vertebrate lineages, including poisonous birds (Pitohui spp. and Ifrita kowaldi; Dumbacher et al, 1992; Dumbacher et al, 2000) and neotropical poison frogs (Phyllobates; Santos et al, 2016), stands out because of its lethality and its unusual ability to facilitate opening and prevent inactivation of voltage-gated sodium channels (NaVs; Catterall, 1977; Khodorov, 1985; Logan et al, 2016; Wang and Wang, 2003). The DIVS6 N→T change reduces BTX sensitivity when tested in rat NaV1.4 (Wang and Wang, 2017), this variant occurs with very low frequency among Phyllobates terribilis (Marquez et al, 2019), and is absent from Phyllobates aurotaenia

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Results

Conclusion