Salmonid fishes are well adapted to transition between salinities as part of a diadromid lifestyle, and many species are both economically and environmentally important. Ion-transporting gill epithelium helps fishes maintain ion balance during salinity transition. Recent transcriptomic surveys suggest that voltage-gated ion channels (VGICs) are present in gill epithelium of fishes. However, fish gill epithelia are architecturally complex and structurally heterogeneous (which includes layers of excitable tissues), which necessitates a model to study isolated gill epithelial cells. In the present study, we isolated gill epithelial cells, used them to reconstruct primary cultured gill epithelium model, and exposed the reconstructed epithelia to apical freshwater (FW). Using RNAseq and molecular biology we demonstrate that multiple VGICs are expressed in cultured gill epithelia of a salmonid, rainbow trout Oncorhynchus mykiss. Following apical exposure to FW, multiple subunits of voltage-gated calcium (Ca