A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na⁺-selective four-domain voltage-gated channels (4D-Na_vs) in animals allowed rapid Na⁺-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na⁺-selective voltage-gated channels (BacNa_v), they typically exhibit much slower kinetics than 4D-Na_vs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na⁺-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) 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, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Na_v blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na⁺-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Na_vs. The EukCatB channels therefore represent an independent evolution of fast Na⁺-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Electrical signals trigger rapid physiological events that underpin an array of fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt, 2015). In excitable animal cells, Ca²⁺- or Na⁺-selective members of the four-domain voltage-gated cation channel family (4D-Ca_v/Na_v) underpin well-characterized signaling processes (Catterall et al., 2017). The 4D-Ca_v/Na_v family is broadly distributed across eukaryotes, contributing to signaling processes associated with motility in some unicellular protist and microalgal species (Fujiu et al., 2009; Lodh et al., 2016), although these channels are absent from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Ca_v/Na_v channel was Ca²⁺-permeable, with Na⁺-selective channels arising later within the animal lineage (Moran et al., 2015). This shift in ion selectivity represented an important innovation in animals, allowing rapid voltage-driven electrical excitability to be decoupled from intracellular Ca²⁺ signaling processes (Moran et al., 2015).Na⁺-selective voltage-gated channels have not been described in other eukaryotes, although a large family of Na⁺-selective channels (BacNa_v) is present in prokaryotes (Ren et al., 2001; Koishi et al., 2004). BacNa_v are single-domain channels that form homotetramers, resembling the four-domain architecture of 4D-Ca_v/Na_v. Studies of BacNa_v channels have provided considerable insight into the mechanisms of gating and selectivity in voltage-dependent ion channels (Payandeh et al., 2012; Zhang et al., 2012). The range of activation and inactivation kinetics of native BacNa_v are generally slower than observed for 4D-Na_v, suggesting that the concatenation and subsequent differentiation of individual pore-forming subunits may have enabled 4D-Na_v to develop specific properties such as fast inactivation, which is mediated by the conserved intracellular Ile–Phe–Met linker between domains III and IV (Fig. 1A; Irie et al., 2010; Catterall et al., 2017).Open in a separate windowFigure 1.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-Na_v (showing the SF of rat 4D-Na_v1.4 with canonical “DEKA” locus of Na⁺-selective 4D-Na_v1s) 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 BacNa_v 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.We recently identified several classes of ion channel (EukCats) bearing similarity to BacNa_v in the genomes of eukaryotic phytoplankton. Characterization of EukCatAs found in marine diatoms demonstrated that these voltage-gated channels are nonselective (exhibiting permeability to both Na⁺ and Ca²⁺) and play a role in depolarization-activated Ca²⁺ signaling (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and EukCatCs) were also identified that remain uncharacterized. These channels are present in haptophytes, pelagophytes, and cryptophytes (EukCatBs), as well as dinoflagellates (EukCatCs; Helliwell et al., 2019). Although there is a degree of sequence similarity between the distinct EukCat clades, the relationships between clades are not well resolved, and there is not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may therefore exhibit significant functional differences. Characterization of these different classes of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel structure, function, and evolution, and to our gaining insight into eukaryote membrane physiology more broadly.Notably, EukCatB channels were found in ecologically important coccolithophores, a group of unicellular haptophyte algae that represent major primary producers in marine ecosystems. Coccolithophores are characterized by their ability to produce a cell covering of ornate calcium carbonate platelets (coccoliths; Fig. 1B; Taylor et al., 2017). The calcification process plays an important role in global carbon cycling, with the sinking of coccoliths representing a major flux of carbon to the deep ocean. Patch-clamp studies of coccolithophores indicate several unusual aspects of membrane physiology, such as an inwardly rectifying Cl⁻ conductance and a large outward H⁺ conductance at positive membrane potentials, which may relate to the increased requirement for pH homeostasis associated with intracellular calcification. Here we report that EukCatB channels from two coccolithophore species (Emiliania huxleyi and Scyphosphaera apsteinii) act as very fast Na⁺-selective voltage-gated channels that exhibit many similarities to the 4D-Na_vs, which underpin neuronal signaling in animals. Thus, our findings demonstrate that the capacity for rapid Na⁺-based signaling has evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.