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. 2020 Oct 1;184(4):1674–1683. doi: 10.1104/pp.20.00889

Figure 3.

Figure 3.

Coccolithophore EukCatB channels are Na+-selective. A, Mean peak currents for coccolithophore EukCatB channels (SaEUKCATB1 and EhEUKCATB1) are shown after substitution of different cations in the external solution. Currents are shown relative to the standard control external solution (E1; Supplemental Table S4). Replacement of extracellular Na+ with NMDG abolished the current in both EukCatB channels. Removal of Ca2+ did not reduce currents for SaEUKCATB1, but greatly reduced those of EhEUKCATB1, which contains two Ca2+-binding EF-hand domains at the C terminus (asterisk indicates 100 μm of EGTA was added to EhEUKCATB1 experiments to ensure consistent removal of Ca2+). Schematic representation of domain architecture of each channel is shown above each graph. CC, Coiled-coil domain (Arrigoni et al., 2016); EF, EF-hand pair; ION, ion transport domain. Error bars: means ± se, n shown in parentheses. Extracellular and intracellular (pipette) solutions are given in Supplemental Table S4. B, Representative currents from A are shown for EhEUKCATB1. C, Mean peak currents after site-directed mutagenesis of the SF of EhEUKCATB1 from TGESWSE to TGDSWSE (E305D). Cation replacement experiments indicate that the EhEUKCATB1 E305D mutant conducts substantial currents in the absence of Na+ (i.e. when Na+ is replaced by NMDG in the external medium). The E305D mutation therefore causes the Na+-selective EhEUKCATB1 channel to become permeable to Ca2+ and Ba2+. Extracellular and intracellular (pipette) solutions are given in Supplemental Table S4, (asteriks indicates 100 μm of EGTA). Mean currents relative to control are shown (Supplemental Table S4). Error bars: means ± se. D, The effect of TTX, a selective Nav blocker, on the mean peak currents of EukCatB channels (E. huxleyi, EhB1; S. apsteinii, SaB1 and SaB2), compared to two EukCatA channels of diatoms (Phaeodactylum tricornutum, PtA1; Odontella sinensis, OsA1 (Helliwell et al., 2019). The effect of TTX on small native Na+ currents in HEK cells is also shown. Mean percent inhibition (relative to untreated control current) is shown, error bars: means ± se; n is given in parentheses.