Evaluating the molecular function of zebrafish Anoctamin 1a

Abstract

Calcium-activated chloride channels (CaCCs) regulate key physiological processes like epithelial secretion, sensory transduction, gastrointestinal pacemaking, and muscle contraction. Zebrafish ( Danio rerio ) are genetically similar to humans, easily manipulated, and are a valuable model organism. We cloned zebrafish Anoctamin 1, a putative CaCC gene, expressed it in mammalian cells, and confirmed its function via halide flux assays. The channel conducted anions and responded to calcium, verifying its role as a CaCC. These findings confirm the functionality of zebrafish CaCCs and lay the groundwork to use the organism to model human physiology and disease.

Figure 1. Heterologous Ano1 expression in CHO-K1 cells

Left: Transfection of plasmids containing (A) vector, (B) mouse Ano1, or (C) zebrafish Ano1. Images were merged and taken at 40X with Ano1 immunoreactivity under the green filter and DAPI staining under the blue filter. Right: (D) Absorbance at 405 nm following a modified Sandell-Kolthoff reaction. Lysates from CHO-K1 cells transfected with empty vector (mean: 0.250, STD: 0.094, n = 6), mouse Ano1 (mean: 0.679, STD: 0.286, n = 6), or zebrafish Ano1 (mean: 0.662, STD: 0.234, n = 6) with the efflux assay run in the presence of 100 µM ionomycin. A Kruskal Wallis test revealed a statistically significant difference in absorbance between groups (H(3) = 10.15, p = 0.002). Dunn’s Post test for multiple comparisons showed significant differences in absorbance between eV and mouse Ano1 (p = 0.01), and between eV and zebrafish Ano1 (p = 0.01). (E) Absorbance at 405 nm following a modified Sandell-Kolthoff reaction. Lysates from CHO-K1 cells transfected with zebrafish Ano1 with the efflux assay run in the presence of 100 µM of ionomycin, or vehicle. An independent T-test showed a significant difference in scores between the two groups, t(10) = 4.16, p = .001. Ionomycin (mean: 0.701, 95% C.I. =0.414 - 0.988, n = 6) had a higher OD405 as compared to the vehicle (mean: 0.230, 95% C.I. = 0.185 - 0.275, n = 6), indicating a calcium dependence of the flux.