marine drugs Article Brevetoxin and Conotoxin Interactions with Single-Domain Voltage-Gated Sodium Channels from a Diatom and Coccolithophore Ping Yates †, Julie A. Koester † and Alison R. Taylor * Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC 28403, USA; [email protected] (P.Y.); [email protected] (J.A.K.) * Correspondence: [email protected] † Authors contributed equally. Abstract: The recently characterized single-domain voltage-gated ion channels from eukaryotic protists (EukCats) provide an array of novel channel proteins upon which to test the pharmacology of both clinically and environmentally relevant marine toxins. Here, we examined the effects of the hydrophilic µ-CTx PIIIA and the lipophilic brevetoxins PbTx-2 and PbTx-3 on heterologously expressed EukCat ion channels from a marine diatom and coccolithophore. Surprisingly, none of the toxins inhibited the peak currents evoked by the two EukCats tested. The lack of homology in the outer pore elements of the channel may disrupt the binding of µ-CTx PIIIA, while major structural differences between mammalian sodium channels and the C-terminal domains of the EukCats may diminish interactions with the brevetoxins. However, all three toxins produced significant negative shifts in the voltage dependence of activation and steady state inactivation, suggesting alternative and state-dependent binding conformations that potentially lead to changes in the excitability of the Citation: Yates, P.; Koester, J.A.; phytoplankton themselves. Taylor, A.R. Brevetoxin and Conotoxin Interactions with Keywords: phytoplankton; membrane excitability; diatom; coccolithophore; algal toxin; brevetoxin; Single-Domain Voltage-Gated µ-conotoxin Sodium Channels from a Diatom and Coccolithophore. Mar. Drugs 2021, 19, 140. https://doi.org/10.3390/ md19030140 1. Introduction Membrane excitability in phytoplankton is facilitated by voltage-gated ion currents Academic Editor: Jean-Marc Sabatier similar to those observed in multicellular organisms [1,2]. Many species encode four- domain (4D), animal-like sodium/calcium channels (Na /Ca ) in addition to the single- Received: 15 January 2021 V V domain (1D) channels recently characterized as the EukCats [3,4]. EukCats are widespread Accepted: 26 February 2021 Published: 2 March 2021 among marine protist groups, including diatoms, coccolithophores and dinoflagellates [3], and contribute to the generation of fast action potentials that play a role in environmental Publisher’s Note: MDPI stays neutral sensing [2,3]. EukCats are homologs of 1D bacterial sodium channels (BacNaVs) [3,5] with regard to jurisdictional claims in that putatively form functional voltage-gated ion channels from homotetramers. Each published maps and institutional affil- 1D monomer of the EukCats contains six transmembrane segments (S1–S6), a voltage- iations. sensing domain (VSD) from S1–S4, with four conserved, positively charged residues on S4, and a selectivity filter (SF) within the pore-loop between S5 and S6. The SF is linked to S5 and S6 by two helices, P1 and P2, respectively (Figure1). Due to the newfound diversity of 1D channels, we are no longer able to predict ion selectivity solely based on the number and identity of negatively charged residues in the primary sequence of amino Copyright: © 2021 by the authors. Bacillus Licensee MDPI, Basel, Switzerland. acids in the seven-residue selectivity filter. For example, the bacterial 1D NaChBac ( + This article is an open access article halodurans) and NaVPp (Plesiocystis pacifica) channels are both Na -selective, but they have distributed under the terms and very different selectivity filters [6,7]. NaChBac contains only a single negatively charged conditions of the Creative Commons glutamic acid (E) in its SF (TLESWAS) [6], whereas NaVPp has three negatively charged Attribution (CC BY) license (https:// residues (TLEDWTD) [7]. Moreover, the prokaryotic calcium channel CaVMr (Meiothermus creativecommons.org/licenses/by/ ruber) has both glutamic and aspartic (D) acids in the SF: TLEGWVD, with the glycine (G) 2+ 4.0/). potentially contributing to Ca -selectivity [7]. Mar. Drugs 2021, 19, 140. https://doi.org/10.3390/md19030140 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, x 2 of 15 Mar. Drugs 2021, 19, 140 (Meiothermus ruber) has both glutamic and aspartic (D) acids in the SF: TLEGWVD,2 with of 15 the glycine (G) potentially contributing to Ca2+‐selectivity [7]. Figure 1. Structure of toxins andand theirtheir hypothesizedhypothesized binding binding sites sites on on one one subunit subunit of of a a single-domain single‐do‐ channel.main channel. (A) upper: (A) upper:µ-CTx μ‐CTx PIIIA PIIIA with with six positively six positively charged charged arginine arginine (R) and(R) and lysine lysine (K) (K) residues resi‐ dues highlighted in blue, from Chen et al. 2014; lower: diagram of a 1D NaV/CaV with the voltage highlighted in blue, from Chen et al. 2014; lower: diagram of a 1D Na /Ca with the voltage sensor sensor of transmembrane segments S1–S4 (grey) and the pore‐domainV (pink)V consisting of S5, the of transmembrane segments S1–S4 (grey) and the pore-domain (pink) consisting of S5, the turret turret loop (yellow), the P1 and P2 helices (blue) with the selectivity filter sequences of EhEUKCATB1loop (yellow), the and P1 OsEUKCATA1, and P2 helices (blue)and S6. with The the red selectivity stars represent filter sequences negatively of charged EhEUKCATB1 acidic resi and‐ duesOsEUKCATA1, aspartic (E) and or glutamic S6. The red(D) starsacid binding represent sites negatively near P1 and charged the selectivity acidic residues filter [8] aspartic and the (E) or glutamicspace between (D) acid P2 binding helices sitesof adjacent near P1 subunits and the selectivitythat μ‐CTx filter PIIIA [8 ]may and theoccupy space when between channels P2 helices are of adjacentinactivated subunits [9]. (B that) upper:µ-CTx PbTx PIIIA‐2; lower: may occupy regions when of PbTx channels binding are inactivatedacross S5, the [9]. turret (B) upper: loop, PbTx-2;and S6 lower:are represented regions of by PbTx the diagonal binding shading across S5, based the turreton homologous loop, and S6regions are represented of binding in by sensitive the diagonal NaVs, NaV1.2, and NaV1.4 [10,11]. shading based on homologous regions of binding in sensitive NaVs, NaV1.2, and NaV1.4 [10,11]. Like bacterial NaVs,s, the the C C-terminus‐terminus of the EukCats contains a coiled-coilcoiled‐coil motif that is hypothesized to be intracellular and perform a rangerange ofof functions,functions, includingincluding stabilizingstabilizing the quaternary structure of the channel [12], [12], or modulating activation [13] [13] and inactiva inactiva-‐ tion [[14]14] of thethe ionion current.current. Bacterial sodiumsodium channelschannels are studiedstudied extensivelyextensively asas modelsmodels for mammalian 4D 4D Na NaVV/Ca/CaVs,V dues, due to their to their natural natural variation variation and andrelative relative ease easeof manipu of ma-‐ nipulationlation [15,16]. [15 The,16]. diversity The diversity of EukCats of EukCats provides provides an expanded an expanded library for library similar for structure similar‐ structure-functionfunction studies with studies the advantage with the advantage of functioning of functioning in eukaryotic in eukaryotic backgrounds backgrounds with dis‐ withtinct physiological distinct physiological and metabolic and metabolic requirements. requirements. Phylogenetically, EukCats group by taxa;taxa; thethe characterizedcharacterized EukCatsEukCats fromfrom diatomsdiatoms and haptophyteshaptophytes (including (including coccolithophores) coccolithophores) form form distinct distinct clades, EukCatAclades, EukCatA and EukCatB, and respectivelyEukCatB, respectively [3]. We focused [3]. We onfocused a EukCatA on a EukCatA from the from diatom the diatomOdontella Odontella sinensis sinensisand a EuKCatBand a EuKCatB from the from coccolithophore the coccolithophoreEmiliania Emiliania huxleyi .huxleyi.Odontella Odontella sinensis sinensisencodes encodes three, single-domainthree, single‐domain EukCatAs EukCatAs [3] that [3] are that functionally are functionally non-selective non‐selective CaVs with CaVs ~10-fold with ~10 faster‐fold + kineticsfaster kinetics than the than Na the-selective Na+‐selective bacterial bacterial channel channel NaChBac NaChBac [3,6]. [3,6]. The selectivityThe selectivity filter filter (on the(on porethe pore loop loop between between S5 and S5 S6) and of S6) OsEUKCATA1, of OsEUKCATA1, from O. from sinensis O. ,sinensis, contains contains two aspartic two acidaspartic residues acid residues (TLDAWAD) (TLDAWAD) that result that in aresult channel in a with channel a similar with permeabilitya similar permeability profile as non-selectiveprofile as non‐ NaChBacselective NaChBac mutants (SF:mutants LEDWAS (SF: LEDWAS and LEDWAD) and LEDWAD) [3,17]. Emiliania [3,17]. Emiliania huxleyi encodeshuxleyi encodes both 4D both (two 4D or three)(two or and three) multiple and EukCatBmultiple isoformsEukCatB (three isoforms or four), (three depending or four), + ondepending the genetic on the strain genetic [2]. EukCatBstrain [2]. channels EukCatB are channels Na -selective, are Na+‐selective, but exhibit but dependency exhibit de‐ 2+ onpendency intracellular on intracellular Ca , which Ca2+ is, which potentially is potentially regulated regulated through through the EF-hand the EF domain‐hand do on‐ themain C-terminus. on the C‐terminus. Their kinetics Their kinetics