Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na⁺/K⁺-ATPase

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na⁺/K⁺-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na⁺/K⁺-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na⁺/K⁺-ATPases of snakes react to the toxins. Some tissues might have Na⁺/K⁺-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na⁺/K⁺-ATPase substitutions and observed resistance using actual snake Na⁺/K⁺-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na⁺/K⁺-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na⁺/K⁺-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa.