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

Abstract

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 (α₁, α₂, α₃) and three β-like isoforms (β₁, β₂, β₄) were identified. The amino acid sequence of α₁-like isoform shared 99.4% identity with the α₁ isoform previously published for R. marina. Sequences for α₂, α₃, and β₄ from R. marina were previously unavailable. The first extracellular loop in the α₂-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 α₃-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 α₁ isoform of Sus scrofa (pig), with α₁-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 α₁ and β₁; skin α₁, β₁, and low levels of β₂; heart α₁, α₃, and β₁; skeletal muscle α₁, β₄, with low levels of α₂, α₃, and β₁. R. marina could be used as an important model for future structural, functional and pharmacological studies of NKA and its isoforms.

Keywords: Cardiac glycosides; Docking; Molecular coevolution; RNAseq; Target site-insensitivity; Toad.