A novel single-domain Na+-selective voltage-gated channel in photosynthetic eukaryotes

The evolution of Na+-selective four-domain voltage-gated channels (4D-Navs) in animals allowed rapid Na+-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signalling. Whilst bacteria encode single-domain Na+-selective voltage-gated channels (BacNav), they typically exhibit much slower kinetics than 4D-Navs, and are not thought to have crossed the prokaryote-eukaryote boundary. As such, the capacity for rapid Na+-selective signalling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus Flytrap where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na+-selective single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and sensitivity to the highly selective 4D-Nav blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na+-based signalling in eukaryotes is not restricted to animals or to the presence of 4D-Navs. The EukCatB channels therefore represent an independent evolution of fast Na+-based electrical signalling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae. One Sentence Summary The capacity for rapid Na+-based signalling has evolved in ecologically important coccolithophore species via a novel class of voltage-gated Na+ channels, EukCatBs.


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Electrical signals trigger rapid physiological events that underpin an array of 53 fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and 54 Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and 55 Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt,56 2015). In excitable animal cells, Ca 2+ -or Na + -selective members of the four-domain voltage-57 gated cation channel family (4D-Cav/Nav) underpin well-characterised signalling processes 58 (Catterall et al., 2017). The 4D-Cav/Nav family is broadly distributed across eukaryotes, 59 contributing to signalling processes associated with motility in some unicellular protist and 60 microalgal species (Fujiu et al., 2009;Lodh et al., 2016), although these channels are absent 61 from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Cav/Nav channel was Ca 2+ 62 permeable, with Na + -selective channels arising later within the animal lineage (Moran et al.,  Figure 1A). 77 We recently identified several classes of ion channel (EukCats) bearing similarity to 78 BacNav in the genomes of eukaryotic phytoplankton. Characterisation of EukCatAs found in 79 marine diatoms demonstrated that these voltage-gated channels are non-selective (exhibiting 80 permeability to both Na + and Ca 2+ ) and play a role in depolarisation-activated Ca 2+ signalling 81 (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and 82 EukCatCs) were also identified that remain uncharacterised. These channels are present in 83 haptophytes, pelagophytes and cryptophytes (EukCatBs), as well as dinoflagellates 84 (EukCatCs) (Helliwell et al., 2019). Although there is a degree of sequence similarity between 85 the distinct EukCat clades, the relationships between clades are not well resolved, and there is 86 not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may 87 therefore exhibit significant functional differences. Characterisation of these different classes 88 of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel 89 structure, function and evolution, and to gain insight into eukaryote membrane physiology 90 more broadly.

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Notably, EukCatB channels were found in ecologically important coccolithophores, a 92 group of unicellular haptophyte algae that represent major primary producers in marine inwardly rectifying Clconductance and a large outward H + conductance at positive membrane 99 potentials, which may relate to the increased requirement for pH homeostasis associated with 100 6 intracellular calcification. Here we report that EukCatB channels from two coccolithophore 101 species (Emiliania huxleyi and Scyphosphaera apsteinii) act as ultra-fast Na + -selective voltage-102 gated channels that exhibit many similarities to the 4D-Navs that underpin neuronal signalling 103 in animals. Thus, our findings demonstrate that the capacity for rapid Na + -based signalling has 104 evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.

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Coccolithophore EukCatBs are fast Na + -selective voltage-gated channels 107 EukCatB sequences are present in haptophyte, cryptophyte and pelagophyte taxa (Helliwell et 108 al., 2019). They form a distinct phylogenetic group from the non-selective Ca 2+ and Na + 109 permeable diatom EukCatAs and prokaryote BacNavs (Figure 1B; Supplementary Table 1). 110 Ion selectivity of voltage-gated channels is mediated by the conserved pore loop region 111 between transmembrane segments S5 and S6, known as the selectivity filter (SF) (Catterall et   To determine the ion transport properties of coccolithophore EukCatB channels, we    Table S4). In contrast, the removal of Ca 2+ from the external solution 143 significanly enhanced the channel conductance in SaEUKCATB1. These data suggest that 144 SaEUKCATB1 represents a fast-activating Na + -selective channel that is entirely distinct from 145 other Na + -selective channels previously described in eukaryotes (4D-Nav).

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The currents generated by EhEUKCATB1 were also dependent on the presence of 147 external Na + . However, EhEUKCATB1 currents were absent in Ca 2+ -free media, suggesting 148 that EhEUKCATB1 may represent a Ca 2+ -dependent Na + channel (Figure 3A and B).

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EhEUKCATB1 possesses a C-terminal Ca 2+ -binding EF-hand pair that may contribute to the 150 observed Ca 2+ -dependent activation. To confirm that the activity of EhEUKCATB1 is Na + -151 dependent, we used site-directed mutagenesis to modify its SF, changing the conserved Glu to 152 Asp (E305D). The EhEUKCATB1-E305D channel was permeable to both Ca 2+ and Ba 2+ in 153 the absence of Na + , indicating that it was no longer Na + -selective ( Figure 3C). This result  We found that 5 µM and 10 µM TTX substantially inhibited EhEUKCATB1 ( Figure 3D), 161 lending strong support to this channel being a Ca 2+ -dependent Na + channel. Although the 162 sensitivity of EhEUKCATB1 to TTX is relatively low compared to the nanomolar sensitivity    Antibiotic Antimycotic (Gibco), and 10% FBS (Gibco) was used to culture cells, which were 275 passaged every 3 to 4 days at 1:6 or 1:12 dilutions (cell/mm 2 ). 277 Sequence similarity searches were carried out as previously outlined by Helliwell     The effect of TTX on small native Na + currents in HEK cells is also shown. Mean % inhibition 451 (relative to untreated control current) is shown, error bars: SEM; n is given in parentheses. to Na + selective Nav1.1, and Ca 2+ permeable Cav2.1 from humans. The coccolithophore 465 sequences most closely resemble the EEEE motif found in metazoan Ca 2+ channels and lack 466 conserved lysines found in Na + channels. The 4D-Cav/Nav sequence found within the S. 467 apsteinii transcriptome represents a partial sequence as it lacks domain I.