Fuelled by creatine: exploring two copies of the creatine transporter SLC6A8 gene in rainbow trout.

The creatine transporter (SLC6A8 or CT1) is essential in creatine metabolism that serves as elementary cellular energy buffer. We identified the two slc6a8 gene variants in the rainbow trout (Oncorhynchus mykiss) genome sharing 69.3% coding sequence similarity. Our analyses indicate that the gene duplication event occurred in a common ancestor of bony fishes, prior to the divergence of tetrapods. While one copy was lost in most vertebrate lineages, both are retained in teleosts. Expression analysis revealed tissue- and cell-specific regulation: both genes were highly expressed in the heart, slc6a8a being predominantly expressed in most tissues and slc6a8b showing substantially elevated expression in muscle and salmonid embryonic CHSE-214 cells. Co-expression patterns with key creatine metabolism enzymes (gatm, gamt, ckm) support a fish-specific regulation of creatine homeostasis. Structural analysis confirmed the presence of 12 transmembrane domains and conserved functionally critical glycosylation motifs in both proteins. In addition, Slc6a8a contained an extracellular amino acid insertion with additional N-glycosylation motif. Fluorescent-labelled Slc6a8a and Slc6a8b proteins were localised to the plasma membrane in CHSE-214 cells, while high-performance liquid chromatography analysis confirmed creatine uptake. This study provides the first characterisation of the duplicated slc6a8 genes in bony fish and suggests their sub-functionalisation during teleost evolution possibly reflecting functional specialisation of the paralogs. We hypothesise that one variant may primarily mediate creatine uptake, whereas the other could contribute to creatine release in distinct tissues under certain physiological conditions.