Identification of Na/K-ATPase α/β isoforms in Rhinella marina tissues by RNAseq and a molecular docking approach at the protein level to evaluate α isoform affinities for bufadienolides.

Na/K-ATPase (NKA) function is inhibited by Bufadienolides (BD), a group of cardiotonic steroids (CTS) primarily produced by anurans of the Bufonidae family, such as Rhinella marina. This study characterized the presence of α and β NKA subunit isoforms in R. marina via RNAseq in four tissues: oocytes, skin, heart, and skeletal muscle. Transcripts encoding three α-like isoforms (α, α, α) and three β-like isoforms (β, β, β) were identified. The amino acid sequence of α-like isoform shared 99.4% identity with the α isoform previously published for R. marina. Sequences for α, α, and β from R. marina were previously unavailable. The first extracellular loop in the α-like isoform in R. marina showed similar substitutions to those found in their susceptible homologues in other taxa (L/Q111T and S119T); in contrast, this same loop in α-like isoform showed similar substitutions (Q111L and G120R) to those reported for toad-eating animals such as snakes, which suggests relatively lower affinity for CTS. Docking results showed that all three α-like isoforms identified in R. marina transcriptomes have low affinity to CTS compared to the susceptible α isoform of Sus scrofa (pig), with α-like isoform being the most resistant. The tissue-specific RNAseq results showed the following expression of NKA α-like and β-like subunit isoforms: Oocytes expressed α and β; skin α, β, and low levels of β; heart α, α, and β; skeletal muscle α, β, with low levels of α, α, and β. R. marina could be used as an important model for future structural, functional and pharmacological studies of NKA and its isoforms.