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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.

Katherine Medina-Ortiz | Diana López-Alvarez | Felipe Navia | Thomas Hansen | Leonardo Fierro | Santiago Castaño
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology | 2021

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.

Pubmed ID: 33476762

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BLASTX (data analysis service)

RRID:SCR_001653

Web application to search protein databases using a translated nucleotide query. Translated BLAST services are useful when trying to find homologous proteins to a nucleotide coding region. Blastx compares translational products of the nucleotide query sequence to a protein database. Because blastx translates the query sequence in all six reading frames and provides combined significance statistics for hits to different frames, it is particularly useful when the reading frame of the query sequence is unknown or it contains errors that may lead to frame shifts or other coding errors. Thus blastx is often the first analysis performed with a newly determined nucleotide sequence and is used extensively in analyzing EST sequences. This search is more sensitive than nucleotide blast since the comparison is performed at the protein level.

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