EukCatBs represent a novel class of single-domain channels. A, Schematic diagram of a single-domain EukCatB channel. The voltage-sensing module (S1–S4, blue), including conserved positively charged (++) residues of segment (S4) that responds to changes in membrane potential, is shown. The pore module (S5–S6, red) is also indicated, including the SF motif (Ren et al., 2001). The structure of a 4D-Nav (showing the SF of rat 4D-Nav1.4 with canonical “DEKA” locus of Na+-selective 4D-Nav1s) is also displayed (right). The Ile–Phe–Met motif of the fast inactivation gate is indicated (West et al., 1992) B, Maximum likelihood phylogenetic tree of single-domain, voltage-gated channels including BacNav and the three distinct classes of EukCat channels (EukCatA–C). Representatives of the specialized family of single-domain Ca2+ channels identified in mammalian sperm (CatSpers) are also included. SF for each sequence is shown (right). “Position 0” of the high-field–strength site that is known to be important in determining Na+ selectivity (Payandeh et al., 2011), is colored red. Channel sequences selected for functional characterization in this study are shown in bold. EukCatA sequences previously characterized (Helliwell et al., 2019) are also indicated, as is NaChBac channel from B. halodurans (Ren et al., 2001). Maximum likelihood bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are above and below nodes, respectively. Scanning electron micrographs of coccolithophores E. huxleyi (scale bar = 2 μm) and S. apsteinii, (scale bar = 10 μm) are shown.