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Role of TRP Channels in Dinoflagellate Mechanotransduction.

Introduction

Mechanosensing, the conversion of mechanical stimuli to chemical and electrical signals, is essential for allowing cells to sense and respond to their physical environment. Numerous mechanosensory pathways involve transient receptor potential (TRP) channels. Of the seven TRP subfamilies (TRPA, TRPV, TRPC, TRPM, TRPML, TRPP, and TRPN), members of TRPV, TRPM, TRPN, TRPP, and TRPC have been described in mechanosensory pathways in animals (Koehler et al., 2006; Yan et al., 2013; Du et al., 2014; Plant, 2014; Shen et al., 2015). Drosophila TRPN is directly sensitive to light touch, while TRPV4 in mammalian endothelial and renal cells is activated by fluid shear stress via a phospholipase A-dependent pathway. TRPC6 is involved in stretch sensing via activation of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and phospholipase C (PLC) in mammalian cardiovascular myocytes (Sharif-Naeini et al., 2010; Yan et al., 2013; Plant, 2014; Mamenko et al., 2015).

Unicellular eukaryotic organisms also possess TRP channels. Bioinformatics studies have revealed TRP channels in the genomes of unicellular organisms including representatives of Choanoflagellata, Chlorophyta, Dictyostelia, Euglenozoa, Ciliophora, and Thecomonadea (Cai and Clapham, 2012; Arias-Darraz et al., 2015a; Peng et al., 2015), indicating that these channels are highly evolutionarily conserved. Yeast Saccaromyces serevisiae TRPY and green alga Chlamydomonas reinhardtii TRP1 are the only cloned and functionally characterized TRP channels in unicellular organisms (Palmer et al., 2001; Arias-Darraz et al., 2015a). TRP11 from C. reinhardtii is important in mechanosensing, leading to changes in swimming pattern in this cell (Fujiu et al., 2011). But overall, there is very limited knowledge of the function of TRP channels in these organisms.

In this study, we investigated the presence and role of TRP channels in the rapid mechanosensing pathway of a unicellular eukaryote, the dinoflagellate Lingulodinium polyedra (F. Stein) J. D. Dodge 1989 (formerly Gonyaulax polyedra; by many authors Lingulodinium polyedrum). Dinoflagellates are members of the Alveolates, protists that belong to the Bikonta (also including, e.g., plants, algae, diatoms, and slime molds), which split from the Unikonta (e.g., animals and fungi) at the root of the eukaryotic evolutionary tree (Stechmann and Cavalier-Smith, 2003).

In nature, mechanical stimulation of the dinoflagellate cell by fluid shear stress or predator contact triggers a flash of light (Rohr et al., 1998; von Dassow et al., 2005). The light display is used as an anti-predator defense behavior and is an important ecological adaptation (Buskey et al., 1983; Mensinger and Case, 1992; Cusick and Widder, 2014). As a reporter of mechanical stress, dinoflagellate bioluminescence is useful as a flow visualization tool (Rohr et al., 1998; Chen et al., 2003; Stokes et al., 2004; Foti et al., 2010).

The bioluminescence mechanotransduction pathway is extremely rapid, with light emission occurring only 15-20 ms after stimulation (Eckert et al., 1965; Widder and Case, 1981; Latz et al., 2008). The signaling pathway involves calcium ([Ca.sup.2+]) flux across the plasma membrane and release of [Ca.sup.2+] from intracellular stores (von Dassow and Latz, 2002). The final step of the signaling pathway is acidification of scintillons due to flux of protons from the vacuole to closely associated scintillons, vesicles containing the luminescent chemistry, resulting in a flash of light (Fogel and Hastings, 1972; Nawata and Sibaoka, 1979; Johnson et al., 1985; Nicolas et al., 1987). The mechanotransduction process occurring at the plasma membrane is currently unknown.

TRP channels are likely candidates for mechanosensing in dinoflagellates because they are evolutionarily conserved, [Ca.sup.2+]-permeable ion channels and are involved in several mechanosensing pathways. Gadolinium ([Gd.sup.3+]) and ruthenium red, frequently used as TRP channel inhibitors (Vriens et al., 2009; Ermakov et al., 2010; Fujiu et al., 2011; Hardie and Franze, 2012), inhibit the mechanically stimulated light response of dinoflagellates (von Dassow and Latz, 2002; Jin et al., 2013).

The specific goals of this study are to identify TRP channels in the dinoflagellate L. polyedra and to determine a putative position for them within the mechanotransduction signaling pathway. In L. polyedra transcriptomic data, we identified proteins with high similarity to mammalian TRPM, TRPP, and TRPML. Agonists and antagonists targeting TRP channels and cellular compounds interacting with TRP in known mechanosensing pathways confirmed their physiological activity in L. polyedra. Results suggest a PLC-dependent signaling pathway, indicating that the role of TRP channels in mechanosensory systems has been conserved through evolution.

Materials and Methods

Bioinformatics

Lingulodinium polyedra transcriptome. We assembled a Lingulodinium polyedra transcriptome from 100-bp paired-end Illumina (San Diego, CA) RNA sequences available at the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/, run accession SRR1184657) using the assembly and annotation pipeline MakeMyTranscriptome (Pierce, 2017), which leverages the Trinity de novo assembler (ver. 2.1.1; Grabherr, 2011). The resulting transcriptome contains 183,451 transcripts, with an N50 of 1161 and 66.5% guanine-cytosine (GC) content. Annotation-based assessment using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed that this transcriptome contained 62% of eukaryotic genes predicted to be present in all eukaryotic assemblies. Open reading frames (ORFs) were predicted using TransDecoder, and the transcriptome was annotated using DIAMOND BLAST-like searches to Swiss-Prot, nr, and UniProt databases. Detailed descriptions of these methods, including all program and database versions used as part of MakeMyTranscriptome, can be found in the supporting information (Appendix S1, available online). The assembled transcriptome has been deposited in Zenodo (Lindstrom et al., 2017).

BLASTP analysis and protein domain identification. In order to identify TRP-like proteins in L. polyedra, we downloaded from https://www.ncbi.nlm.nih.gov/ more than 370 TRP amino acid (AA) sequences, including members of mammalian TRPA, TRPV, TRPM, TRPC, TRPML, TRPP, Drosophila sp. TRP and TRPL, and Caenorhabditis elegans OSM-9, as well as sequences without annotation on subfamily. We used the downloaded sequences as query sequences in BLASTP searches of the L. polyedra transcriptome ORF database using Geneious bioinformatics software, version 9.1.3 (Biomatters, Auckland, New Zealand).

TRP channels are highly diverse, both between species and between subfamilies within species, but they can be identified by a conserved six-transmembrane domain, constituting the cation-permeable ion pore and shared between all described TRP channels. TRPC, TRPM, and TRPN also share the TRP domain, a 23-25 AA conserved sequence upstream of the six-transmembrane domain (Venkatachalam and Montell, 2007; Latorre et al., 2009).

We selected L. polyedra AA sequences with e < [10.sup.-15] and coverage > 40% to TRP query proteins for transmembrane structure analysis using Hidden Markov Model (HMM) (Geneious, ver. 9.3.1) and transmembrane helix prediction (TMHMM) (Krogh et al., 2001). Furthermore, in order to identify putative L. polyedra TRP domains, we aligned selected L. polyedra AA sequences with mammalian TRPC1-7 and TRPM1-8 and Drosophila TRP, TRPM, TRP[gamma], and TRPL as well as Danio rerio TRPN1 and C. elegans TRPN (Venkatachalam and Montell, 2007) TRP domain sequences, using MUSCLE multiple alignment (Edgar, 2004) (Geneious, ver. 9.3.1).

Phylogenomics. We used MAFFT alignment, version 7 (Katoh et al., 2002; Katoh and Standley, 2013), to align selected L. polyedra AA sequences with published TRP channel sequences representing all the human TRP subfamilies, as well as TRP subfamilies from Danio rerio (Dr), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Monosiga brevicollis (Mb), Paramecium tetraurelia (Pt), and Chlamydomonas reinhardtii (Cr). Thereafter, we trimmed the alignment manually to contain the evolutionarily conserved TRP six-transmembrane domain of all included sequences, as predicted by HMM (Peng et al., 2015). A phylogenetic tree was inferred using computer-generated imagery (http://www.phylogeny.fr/index.cgi) with the following pipeline: Maximum likelihood method PhyML, version 3.1/3.0, substitution model WAG, bootstrap value of 100; and the tree was constructed using TreeDyn, version 198.3 (Dereeper et al., 2008). The tree was edited using FigTree, version 1.4.3 (Rambaut, 2016).

We collected the sequences included in the phylogenetic tree from http://www.jgi.doe.gov (Mb sequences) and http://www.ncbi.nlm.nih.gov/protein (all other sequences). Accession numbers are available in Table Al.

Pharmacology

Cell cultures. We cultured L. polyedra strain CCMP1932 in half-strength f/2 growth medium (Guillard and Ryther, 1962) prepared from aged GF/F (GE Whatman, Pittsburgh, PA) filtered seawater (FSW) collected from Scripps pier (La Jolla, CA). We maintained cultures in an environmental chamber at 19-22 [degrees]C with cool white illumination on a light/dark cycle of 12h : 12 h and used them for pharmacological experiments during their exponential growth phase.

The bioluminescence of L. polyedra has a diurnal rhythm, with light capacity and excitability that are low during the light phase and high during the dark phase (Biggley et al., 1969; Fritz et al., 1990). We prepared samples of L. polyedra cell culture at the end of the light phase and estimated the cell concentration, expressed as cells [ml.sup.-], by counting subsamples under a stereomicroscope. We used cultures at concentrations of 3000-7000 cells [ml.sup.-1] and aliquoted 0.5-1.0-ml samples into 7-ml glass scintillation vials that remained in darkness until the start of the experiment, 3-4 h into the dark phase when the cells had reached full bioluminescence capacity (Biggley et al., 1969).

Chemicals. We selected compounds for testing based on their reported effects on TRP-mediated and mechanosensing signaling pathways (Table 1 and below). AMTB hydrochloride (N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride), arachidonic acid, capsaicin, capsazepine, carvacrol, gadolinium chloride ([Gd.sup.+]), HC067047, RHC80267, ML204, and U73122 were purchased from Sigma Aldrich (St. Louis, MO). Tarantula Grammostola spatulata mechanotoxin 4 (GsMTx4) and RN1747 were obtained from Tocris Bioscience (Minneapolis, MN); SKF 96365 hydrochloride from Abcam (Cambridge, MA); and acetic acid and dimethyl sulfoxide (DMSO) from Thermo Fisher Scientific (Waltham, MA).

We prepared acetic acid (1 mol [1.sup.-1]) in Milli-Q water (MilliporeSigma, Burlington, MA) and stock solutions of [Gd.sup.3+] in FSW. All other chemicals were dissolved in DMSO prior to preparation of working solutions in FSW. The final concentration of DMSO was <0.5%. The final concentrations of 10 [micro]mol [1.sup.-1] RN1747 and arachidonic acid are approximate because the DMSO-dissolved stock solution dissolved poorly in FSW.

The TRPM inhibitor AMTB hydrochloride (Lashinger et al., 2008; Quallo et al., 2015), the TRPV inhibitor RN1734 (Vincent et al., 2009; Vriens et al., 2009; Bagher et al., 2012), the TRPV and TRPM inhibitors HC067047 (Atala, 2011; Miyamoto et al., 2014) and capsazepine (Bevan et al., 1992; Docherty et al., 1997; Behrendt et al., 2004; Vriens et al., 2009; Olszewska and Tegowska, 2011; Zhang et al., 2015b), the TRPV and TRPC inhibitor SKF 96365 (Li et al., 2005; Kurth et al., 2015), and the general mechanosensing ion channel inhibitor GsMTx4 (Suchyna et al., 2004; Hurst et al., 2009; Bae et al., 2011) were included in an initial screen. These compounds were not used in further experiments due to toxicity issues or inconclusive effects on bioluminescence, briefly mentioned in Results.

Light recording--photomultiplier. We measured the light responses of L. polyedra to chemical and mechanical treatments as previously described (Jin et al., 2013). Briefly, we measured the samples in a 15-cm-diameter light-integrating chamber (Labsphere, North Sutton, NH) using a photon-counting photomultiplier detector (PMT; Electron Tubes model P30232, ET Enterprises, Uxbridge, UK) fitted with a Uniblitz electronic shutter (Vincent Associates, Falmouth, MA). We placed a neutral-density filter (ND1.0, Kodak Wratten #96, Eastman Kodak, Rochester, NY) between the sample and the PMT detector to avoid light saturation of the detector, and we collected data using 10-ms integration, expressed as photons per second, based on prior light calibration.

Experimental setup. We performed three types of pharmacological experiments: (1) inhibition by chemical pretreatment (antagonist) followed by stimulation by physical treatment (stirring); (2) pretreatment with antagonist followed by stimulation by chemical treatment (agonist); and (3) stimulation by agonist.

In experiment types 1 and 2, we manually and slowly added 0.05-0.1 -ml antagonist or control pretreatment solution by pipette to the test vials 1 h prior to the start of the experiments. Following incubation, we carefully inserted the vials into the light-integrating chamber and stimulated either with agonist or by stirring at a constant speed (Jin et al., 2013). In experiment type 1, we began stirring at the start of the measurement and measured the response for 1 min. In experiment type 2, we added 0.1-0.5-ml agonist or control solution to the sample at the start of the measurement using a syringe pump (Harvard PHD2000, Harvard Apparatus, Holliston, MA, or KDS230, KD Scientific, Holliston, MA) at 1 ml [min.sup.-1] and measured the light response for 5 min. We performed experiment type 3 as for experiment type 2 but omitted the pretreatment step. We altered the order of testing (e.g., control, treatment 1, treatment 2; control, treatment 1, treatment 2, etc.) to avoid bias due to possible temporal trends in the luminescent capacity of the cells (Biggley et al., 1969; Fritz et al., 1990).

Results for each sample are expressed in photons per second per cell as the average light response during the time of measurement (1 or 5 min). Light stimulation during the addition of the agonist or control solution was not different between treatments in any experiment (data not shown); this period of measurement was omitted from the data analysis.

We used measurement of total luminescence capacity by the acidification of the cells to indicate potential toxicity of the compounds used (von Dassow and Latz, 2002; Jin et al., 2013). A 250-[micro]l volume of 1 mol [1.sup.-l] acetic acid was added to a test vial, either following 1 h of pretreatment with antagonist or following agonist treatment. The resulting pH in the samples after the addition of acetic acid was <3, sufficient to activate the bioluminescence chemistry independent of up-stream physiological mechanisms (Hastings and Sweeney, 1957; Fogel and Hastings, 1972; Sweeney, 1986). The response to acidification was measured using a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany) or a PMT (described above). Total luminescence capacity is the combined light emitted from the chemical stimulation and acidification of cells. Concentrations of compounds resulting in a lower total luminescence capacity that was significantly different from the control were considered toxic to the cells and were not used for further experiments.

Statistical analysis. Values are presented as average [+ or -] standard error unless otherwise stated. We replicated all experiments at least twice, on separate days and with different cultures, unless otherwise stated. Prior to statistical analysis, we normalized each value to the average of the control and treatment data in one sample set (see Experimental setup above). Data from capsaicin type 3 experiments (see Experimental setup above) were not normalized. Furthermore, we analyzed effects of treatments using a mixed-model ANOVA with pretreatment and treatment as fixed factors and date as a random factor, followed by a Student-Newman-Keuls post hoc test using SPSS statistical software (IBM SPSS Statistics 24; Armonk, NY). We used Cochran's test for homogenous variances prior to the ANOVA (Cochran, 1941). Only the data derived from testing the effect of [Gd.sup.3+] (5 [micro]mol [1.sup.-1]) on capsaicin-stimulated luminescence had nonhomogenous variances, and thus, effects of treatments were tested using the Kruskal-Wallis nonparametric test (IBM SPSS Statistics 24). A P-value [less than or equal to]0.05 indicated statistical significance. The mixed-model ANOVA revealed a difference in response between dates for 5 and 10 [micro]mol [1.sup.-1] RN1747 and arachidonic acid, as well as 10 [micro]mol [1.sup.-1] RHC80267. Because these differences did not affect the differences between treatments, which were similar whether combining the separate experiments or analyzing single experiments separately, they are not indicated in Figures 3 and 4.

Results

Bioinformatics

There was high sequence similarity between several query TRP proteins and proteins in the Lingulodinium polyedra transcriptome ORF database. Top BLASTP hits were typically around 500 AA and mapped primarily to the conserved six-transmembrane ion pore domain or to the N-terminal part of the TRP query proteins. The highest similarities and coverage were found with TRPM, TRPP, and TRPML query sequences. Shorter L. polyedra AA sequences with high similarity to TRPA and TRPV were also identified. However, as they align with ankyrin repeat domains, common in TRPA and TRPV as well as many other types of proteins (Sedgwick and Smerdon, 1999; Mosavi et al., 2004; Venkatachalam and Montell, 2007), we could not determine with confidence whether these sequences were related to TRP channels; so they were not considered for further analyses.

BLASTP analysis identified TRPM, TRPP, and TRPML-like proteins in Lingulodinium polyedra. We identified six L. polyedra TRP-like AA sequences in the ORF database of the L. polyedra transcriptome (Table 2). Lp1, Lp2, Lp3, Lp4, and Lp5 had e< [10.sup.-15] and coverage >40% to mammalian TRPM2 (Lp1), TRPM8 (Lp2), TRPP1 (Lp3), and TRPP2 (Lp4 and Lp5), as well as Xenopus laevis TRPM8 (Lp2). Lp6 had e < [10.sup.-15] and 39% coverage to human TRPML2. We selected these sequences for further analysis of protein transmembrane structures and conserved domains. The Lp1-Lp6 sequences are deposited in the Zenodo open research repository (see Lindstrom et al., 2017).

Six-transmembrane domain and TRP domain prediction. The transmembrane helix analysis (TMHMM) predicts six transmembrane helices in Lp3, Lp4, and Lp5 and four transmembrane helices for Lp6. However, HMM predicts 6 transmembrane structures with 100%, 100%, 60%, 60%, 100%, and 100% certainty for Lp6 (Fig. 1 A, B), as well as a possible seventh transmembrane structure with 50% certainty, positioned between structures 5 and 6 (not shown). The TMHMM and HMM analyses also show a longer AA distance between helix 1 and helix 2 compared to the distance between the other helices in the six-transmembrane structure in Lp3-Lp6. The longer AA distance is characteristic for TRPML and TRPP in other organisms (Venkatachalam and Montell, 2007).

In Lp1 and Lp2, the TMHMM analysis predicts five and three transmembrane helices, respectively, while the HMM analysis reveals six putative transmembrane structures for both sequences (Fig. 1A, B). In both Lp1 and Lp2, the two most c-terminal helices (5 and 6) are predicted with 100% certainty. These regions form the ion permeation pathway in characterized TRP proteins (Venkatachalam and Montell, 2007; Latorre et al., 2009; Li et al., 2011; Voets, 2012). Details of the HMM analysis are provided in Figure A1.

We identified the TRP domain, shared between mammalian TRPM and TRPC as well as Caenorhabditis elegans TRPN, Danio rerio TRPN, Drosophila TRPM, TRP[gamma], and TRPL, and Chlamydomonas reinhardtii TRP1 (Venkatachalam and Montell, 2007; Latorre et al., 2009; Arias-Darraz et al., 2015a) in Lp1 and Lp2, but not in Lp3-Lp6, when aligning Lp1 and Lp2 with mammalian TRPM1-TRPM8 TRP domain sequences (Fig. 1C) but not when using mammalian TRPC1-TRPC7, Drosophila TRP, or D. rerio or C. elegans domain sequences in the alignment.

The L. polyedra TRP domain is located in the c-terminal part of the protein, downstream from the ion channel domain (Fig. 1A) at AA 1003-1025 for Lp1 and at AA 976-1001 for Lp2. We also identified TRP boxes 1 and 2, both 5-AA highly conserved parts of TRP domains in Lp1 and Lp2 (Fig. 1C). Tryptophan on position 1 in TRP box 1 is the most conserved AA of the TRP domain and is shared with Lp1 and Lp2. Positively charged AA on positions 2 and 5 are also highly conserved and suggested to interact with phosphatidylinositol 4,5-bisphosphate ([PIP.sup.2]) in mammalian TRP (Latorre et al, 2009; Arias-Darraz et al., 2015a). Lp1 and Lp2 have a lysine on position 5 but lack a positively charged AA on position 2 (Fig. 1C).

TRP phylogeny. Phylogenetic inference of the six-transmembrane domain including Lp1-Lp6 and selected TRP family members from Homo sapiens (Hs), Danio rerio (Dr), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Monosiga brevicollis (Mb), Paramecium tetraurelia (Pt), and Chlamydomonas reinhardtii (Cr) places Lp1 and Lp2 in the TRPM clade, Lp3-Lp5 in the TRPP clade, and Lp6 in the TRPML clade (Fig. 2).

Pharmacology

TRP channel agonists stimulate bioluminescence in Lingulodinium polyedra. Three TRP channel agonists, capsaicin, RN1747, and arachidonic acid, representing at least two different mechanisms of TRP channel activation, all stimulated the light response in L. polyedra in a dose-dependent manner (Fig. 3). Capsaicin, an activator of mammalian TRPV1 (Vriens et al., 2009; Cao et al., 2013) and TRP-related responses in insects and annelids (Olszewska and Tegowska, 2011; Summers et al., 2014) at a concentration of 10 [micro]mol [1.sup.-1] caused 3 times the light emission (1.83 [+ or -] 0.27 photons [s.sup.-1] [cell.sup.-l]) compared to controls (0.59 [+ or -] 0.09 photons [s.sup.-1] [cell.sup.-1]; Fig. 3A; Video 1, available online), a result that was significantly different ([n.sub.control] = 24, [n.sub.capsaicin] = 16, P = 0.008). Moreover, the light response of L. polyedra was stimulated by RN1747, an activator of mammalian TRPV4 channels with a yet-unknown mechanism of action (Vincent et al., 2009; Vincent and Duncton, 2011). At a concentration of 5 [micro]mol [1.sup.-1] RN1747, the light response of 3.60 [+ or -] 0.62 photons [s.sup.-1] [cell.sup.-1] was 4 times higher than controls (0.91 [+ or -] 0.13 photons [s.sup.-1] [cell.sup.-1]), representing a significant difference ([n.sub.control] = 16, [n.sub.RN1747] = 16. P = 0.01; Fig. 3B).

Arachidonic acid and its breakdown products and/or compounds, including arachidonic acid, stimulate mammalian TRPC6, several isoforms of mammalian TRPV and TRPM, as well as Drosophila TRP and TRPL channels (Chyb et al., 1999; Watanabe et ai, 2003; Aires et al., 2007; Meves, 2008). In this study, arachidonic acid greatly stimulated the light response in L. polyedra. A concentration of 5 [micro]mol [1.sup.-1]' resulted in light emission of 6.67 [+ or -] 0.58 photons [s.sup.-1] [cell.sup.-1], 11 times higher than the control (0.61 [+ or -] 0.07 photons [s.sup.-1] [cell.sup.-1]), representing a significant difference ([n.sub.control] = 20, [n.sub.arachidonic acid] = 20, P = 0.007; Fig. 3C). Higher concentrations of capsaicin, RN1747, and arachidonic acid resulted in even higher light responses (Fig. 3).

Antagonists of TRP-mediated signaling pathways inhibit the mechanically stimulated light response. We screened several TRP channel antagonists for their inhibiting effect on the mechanically stimulated light response in L. polyedra, including compounds directed against mammalian TRP subfamilies TRPV, TRPM, and TRPC, as well as general inhibitors of mechanosensitive ion channels (Table 1).

The TRPC channel antagonist ML204 at a concentration of 1 [micro]mol [1.sup.-1] decreased the average light response to stirring stimulation by 24% (91.4 [+ or -] 4.37 photons [s.sup.-1] [cell.sup.-1]) compared to the control (119.9 [+ or -] 2.63 photons [s.sup.-1] [cell.sup.-1]), a result that was significantly different ([n.sub.Control] = 15, [n.sub.ML204] = 15, P = 0.043; Fig. 4A).

The mechanosensitive ion channel inhibitor [Gd.sup.3+] inhibits mechanically stimulated bioluminescence in L. polyedra at a concentration of 200 [micro]mol [1.sup.-1] (Jin et al., 2013). In this study, a much lower concentration of 5 [micro]mol [1.sup.-1] [Gd.sup.3+] inhibited the light response to stirring stimulation by 47% (60.0 [+ or -] 5.93 photons [s.sup.-1] [cell.sup.-1]) as compared to the control (113.7 [+ or -] 5.93 photons [s.sup.-1] [cell.sup.-1], [n.sub.control] = 6, [mathematical expression not reproducible] = 6, P < 0.0001; Fig. 4B).

The PLC inhibitor U73122 and the DAG-lipase inhibitor RHC80267 also inhibited the stirring-stimulated light response. U73122 at a concentration of 1 [micro]mol [1.sup.-1] inhibited the response by 33% (68.5 [+ or -] 4.63 photons [s.sup.-1] [cell.sup.-1]) as compared to thecontrol(103.0[+ or -]4.0 photons [s.sup.-1] [cell.sup.-1], [n.sub.Control] = 30, [n.sub.U73l22] = 29, P = 0.01; Fig. 4C); and RHC80267 at a concentration of 10 [micro]mol [1.sup.-1] inhibited the response by 29% (73.3 [+ or -] 3.71 photons [s.sup.-1] [cell.sup.-1]) as compared to the control (103.9 [+ or -] 5.17 photons [s.sup.-1] [cell.sup.-1] , [n.sub.control] = 28, [n.sub.73122] = 28, P< 0.0001; Fig. 4D).

Carvacrol, which inhibits mammalian TRPM and Drosophila TRPL (Parnas et al., 2009; Chen et al., 2015), had no significant effect on the stirring-induced light response in L. polyedra. At the highest tested concentration of 500 [micro]mol [1.sup.-1], the response to stirring was 105.0 [+ or -] 4.42 photons [s.sup.-1] [cell.sup.-1] after pretreatment with carvacrol, as compared to 97.7 [+ or -] 12.52 photons [s.sup.-1] [cell.sup.-1] in the control treatment ([n.sub.control] = 5, [n.sub.carvacrol] = 5, P = 0.461; Fig. 4E).

We also screened the effect of the TRPV inhibitor RN1734, the TRPV and TRPM inhibitor HC067047, the TRPV and TRPC inhibitor SKF 96365, and the general mechanosensing ion channel inhibitor GsMTx4 on stirring-induced luminescence. These compounds were found to be toxic to the cells in low concentrations; therefore, we did not use them for further experiments. The TRPV and TRPM inhibitor capsazepine and the TRPM inhibitor AMTB hydrochloride had stimulating effects on bioluminescence per se and were also omitted from further experiments.

Antagonists of TRP-mediated signaling pathways did not inhibit the capsaicin-stimulated light response. Concentrations of ML204, [Gd.sup.3+], and U73122 that inhibited the mechanically stimulated light response, as described above, did not inhibit the capsaicin-stimulated light response (Fig. 5). ML204 even had a potentiating effect on the capsaicin response (Fig. 5A).

Pretreatment with the mammalian TRPC antagonist ML204 at a concentration of 1 [micro]mol [1.sup.-1] potentiated the response to 30 [micro]mol [1.sup.-1] capsaicin, with a 34% higher response (13.3 [+ or -] 0.70 photons [s.sup.-1] [cell.sup.-1]) compared to the capsaicin control (9.8 [+ or -] 0.41 photons [s.sup.-1] [cell.sup.-1]), representing a significant difference ([n.sub.all treatments] = 7, P < 0.0001; Fig. 5A).

Pretreatment with [Gd.sup.3+] at 5 [micro]mol [1.sup.-1] had no significant effect on the response to 30 [micro]mol [1.sup.-1] capsaicin (9.9 [+ or -] 0.62 photons [s.sup.-1] [cell.sup.-1]) compared to the capsaicin control (9.3 [+ or -] 0.36 photons [s.sup.-1] [cell.sup.-1], [n.sub.all treatments] = 12, P = 0.419; Fig. 5B). However, we observed that higher concentrations of [Gd.sup.3+] potentiated the capsaicin-stimulated light response, although the magnitude of this effect varied among experiments. In one experiment using [Gd.sup.3+] purchased from Tocris, the response to 30 [micro]mol [1.sup.-1] capsaicin after 14 [micro]mol [1.sup.-1] [Gd.sup.3+] pretreatment was 25.2 [+ or -] 0.93 photons [s.sup.-1] [cell.sup.-1], 110% higher than the capsaicin control of 12.0 [+ or -] 0.60 photons [s.sup.-1] [cell.sup.-1], representing a significant difference ([n.sub.all treatments] = 6, P < 0.0001). In two combined experiments using [Gd.sup.3+] from Sigma Aldrich, the response to 30 [micro]mol [1.sup.-1] capsaicin after 20 [micro]mol [1.sup.-1] [Gd.sup.3+] pretreatment was 26% higher (11.6 [+ or -] 1.07 photons [s.sup.-1] [cell.sup.-1]) compared to the capsaicin control (9.2 [+ or -] 0.36 photons [s.sup.-1] [cell.sup.-1]), although this was not a significant difference ([n.sub.all treatments] = 12, P = 0.074).

Pretreatment with the PLC inhibitor U73122 at a concentration of 1 [micro]mol [1.sup.-1] did not inhibit the light response to 30 [micro]mol [1.sup.-1] capsaicin (13.0 [+ or -] 1.02 photons [s.sup.-1] [cell.sup.-1]) compared to capsaicin controls (11.5 [+ or -] 0.56, [n.sub.all treatments] = 12, P = 0.423; Fig. 5C).

Discussion

The ability of cells to sense their physical environment is an ancient process that involves mechanotransduction signaling pathways containing mechanically activated ion channels. Prokaryotic organisms possess stretch-activated ion channels that regulate cell turgor for osmoregulation. Eukaryotic cells have sophisticated capabilities for sensing not only stretch but also environmental mechanical stress including tensile forces, compressive forces, and sound waves. The diversity of sensory stimuli reflects the expression of a range of specialized ion channels involved directly or indirectly in mechanosensing signaling pathways (Syntichaki and Tavernarakis, 2004; Martinac et al., 2008; Arnadottir and Chalfie, 2010; Delmas et al., 2011; Haswell et al., 2011; Katta et al., 2015). In this study, we find both sequence homology and pharmacological support for the presence of TRP-like channels in a eukaryotic alveolate protist, and our results are consistent with their role in rapid mechanosensing as a part of predator avoidance behavior.

BLASTP and phylogenetic inference indicate that Lingulodinium polyedra expresses TRPM (Lp1 and Lp2)-like, TRPP (Lp3-Lp5)-like, and TRPML (Lp6)-like proteins. The TRPP and TRPML similarity of Lp3-Lp5 and Lp6, respectively, is further supported by the 145-248-AA distance between transmembrane helices 1 and 2, as predicted with TMHMM and HMM. This long distance is characteristic of the TRPP and TRPML subfamilies (Christensen and Corey, 2007; Venkatachalam and Montell, 2007). Moreover, the presence of the TRP domain, common for TRPM, TRPC, and TRPN (Venkatachalam and Montell, 2007) in Lp1 and Lp2, further supports their similarity to TRPM.

There are few reports of TRPM channels in unicellular organisms. TRPP, TRPML, and TRPV have been proposed as the ancestors of metazoan TRP channel subfamilies, based on the presence of these channels and lack of homologs to other metazoan TRP subfamilies in unicellular eukaryotes, including the apusozoan Thecamonas trahens (expressing TRPP and TRPV) and the amoebozoan Dictyostelium (expressing TRPP and TRPML), both located near the base of the unikont branch of the evolutionary tree. Only TRPP and TRPML have been reported in Bikonta species of the euglenozoans Leishmania and Trypanosoma and the alveolate protist Paramecium (Cai and Clapham, 2012; Arias-Darraz et al., 2015a; Peng et al., 2015). In the bikont unicellular green alga Chlamydomonas reinhardtii, Arias-Darraz et al. (2015b) report the presence of TRPM-like sequences in the proteome of C. reinhardtii, while Arias-Darraz et al. (2015a) identify the TRP domain in TRP1, suggesting relatedness of TRP1 to TRPM, TRPC, or TRPN. In phylogenetic inference (Arias-Darraz et al., 2015a; this study), C. reinhardtii TRP1 is located within the TRPM-, TRPC-, and TRPN-branch but separate from the specific branches of those subfamilies (Arias-Darraz et al., 2015a; Peng et al., 2015), while the TRPM-like Lp1 and Lp2 in this study are located within the TRPM branch. Other studies suggest that TRPM, along with TRPA and TRPC, has evolved later in the unikont lineage, as it is present in the genomes of the choanoflagellates Monosiga brevicollis and Salpingosea rosetta, representing the last unicellular ancestor of Metazoa (Mederos y Schnitzler et al., 2007; Cai, 2008; Cai and Clapham, 2012; Peng et al., 2015). The presence of TRPM-like proteins in L. polyedra suggests that TRPM, together with TRPV, TRPP, and TRPML, was present in a common ancestor of Unikonta and Bikonta.

All identified TRP-like proteins in L. polyedra have the potential to be involved in mechanosensing; channels belonging to the TRPM and TRPP families are described in shear stress and stretch-sensing signaling pathways of mammalian endothelial cells and vascular smooth muscle cells, as well as in sensing of light touch in Drosophila larvae (Earley et al., 2004; Nauli et al., 2008; AbouAlaiwi et al., 2009; Plant, 2014; Turner et al., 2016). Caenorhabditis elegans TRPP homologs LOV-1 and PKD-2 are expressed in sensory cilia and are essential for mating success between worm hermaphrodites (Barr and Sternberg, 1999; Kahn-Kirby and Bargmann, 2006). Human TRPP 1 and TRPP2 are involved in flow sensing in renal cells; malfunction of these channels results in polycystic kidney disease, manifesting as fluid-filled cysts in the tissue (Nauli et al., 2003; Kahn-Kirby and Bargmann, 2006). TRPML is rarely described in the context of mechanosensing. However, TRPML3 is abundantly expressed in mouse ear hair cells and may be involved in mechanosensing related to hearing, although this conclusion is controversial (Di Palma et al., 2002; van Aken et al., 2008; Wu et al., 2016).

In this study, the TRP channel agonists capsaicin, RN1747, and arachidonic acid stimulated bioluminescence in L. polyedra. These results are consistent with previous results showing that ruthenium red and [Gd.sup.3+] inhibit mechanically stimulated bioluminescence (von Dassow and Latz, 2002; Jin et al., 2013); the results also indicate that TRP channels have an important role in the L. polyedra mechanosensing pathway. Ruthenium red also inhibits [Ca.sup.2+] release from intracellular stores (Chamberlain et al., 1984; von Dassow and Latz, 2002; Jain and Sharma, 2016), and both ruthenium red and [Gd.sup.3+] inhibit some voltage-gated [Ca.sup.2+] channels (Lacampagne et al., 1994; Malecot et al., 1998), as well as Piezo channels, a recently discovered mechanosensitive ion channel conserved from protozoa to mammals (Coste et al., 2010, 2012; Prole and Taylor, 2013; Volkers et al., 2015). Therefore, the effects of the more TRP-specific compounds capsaicin and RN1747 provide strong evidence that a TRP channel is involved in L. polyedra mechanosensing. Arachidonic acid was the most effective activator of luminescence in this study. Arachidonic acid is a polyunsaturated fatty acid natively produced in many cells including dinoflagellates; it acts as an intrinsic second messenger in many cellular processes (Meves, 2008; Fuentes-Grunewald et al., 2012). Arachidonic acid can interact with TRP channels incorporated in the DAG molecule, as free arachidonic acid or processed to some of its breakdown products, the epoxyeicosantrienoic acids (EET) (Chyb et al., 1999; Watanabe et al., 2003; Oike et al., 2006; Aires et al., 2007). In this study, inhibition of mechanically stimulated luminescence in L. polyedra with the DAG-lipase inhibitor RHC80267 suggests that a DAG breakdown product, for example, arachidonic acid, can activate TRP channels.

Our choice of agonists mainly targeted TRPV channels, because they are frequently described in mechanosensing pathways in mammals and because mechanosensing in the unicellular algae C. reinhardtii is dependent on TRP11, a TRPV-type channel (Fujiu et al., 2011; Plant, 2014). However, even as luminescence was stimulated using TRPV agonists in this study, none of the TRPV-specific antagonists screened could be used to physiologically inhibit mechanically stimulated bioluminescence in L. polyedra. SB-366791, RN1734, and HC067047 were all toxic to the cells already at a concentration of 1 [micro]mol [1.sup.-1], while 5 [micro]mol [1.sup.-1] capsazepine stimulated light production. Capsazepine is a competitive antagonist to capsaicin; stimulation of the light response using this compound suggests a physiological interaction with an L. polyedra TRP-like channel, though it was not inhibiting. Similarly, AMTB, an inhibitor of mammalian TRPM8, stimulated luminescence, also suggesting an interaction with L. polyedra TRP-like channels. On the other hand, carvacrol, an inhibitor of mammalian TRPM7 channels, had no effect on stirring-induced luminescence or stimulated luminescence. Most available pharmacological compounds used with TRP channels are manufactured, optimized, and tested for specific isoforms of mammalian TRP channels and cannot be assumed to interact with the same specificity with dinoflagellate TRP-like ion channels.

Unexpectedly, ML204 potentiated the capsaicin-stimulated luminescence even though it reduced the stirring-induced luminescence. ML204 is a recently developed compound with an unknown mechanism of action that has not been widely used (Miller et al., 2011; Westlund et al., 2014; Wei et al., 2015). Therefore, it is difficult to speculate on the different effects of this compound on stirring- and capsaicin-induced luminescence in L. polyedra. [Gd.sup.3+] at a concentration of 5 [micro]mol [1.sup.-1] had no effect on the capsaicin-induced luminescence, while a potentiating effect was observed in some experiments for higher concentrations of [Gd.sup.3+]. Considering the many reported cellular targets of [Gd.sup.3+], it is possible that [Gd.sup.3+] inhibits stirring-induced luminescence in this study by another mechanism than through inhibition of TRP channels (Lacampagne et al., 1994; Coste et al., 2010; Ermakov et al., 2010; Volkers et al., 2015). Increased membrane fluidity stimulates luminescence in L. polyedra, while the voltage-gated [Ca.sup.2+] channel blocker nifedipine inhibits the light response (Mallipattu et al., 2002; Jin et al., 2013). Therefore, the action of [Gd.sup.3+] may be through the reduction of plasma membrane fluidity or the inhibition of voltage-gated [Ca.sup.2+] channels. Similar to our results, [Gd.sup.3+] potentiates capsaicin-stimulated responses in mammalian TRPV 1 (Tousova et al., 2005). The different effects of [Gd.sup.3+] in this study suggest that [Gd.sup.3+] is targeting different parts of the luminescence-stimulating mechanosensing pathway in L. polyedra.

The inhibitory effect of PLC-antagonist U73122 on mechanically stimulated luminescence but not on capsaicin-stimulated luminescence suggests that TRP channels are not the primary mechanotransducer in the L. polyedra mechanosensing pathway. This conclusion is in agreement with several mechanosensing and other TRP-mediated pathways in other organisms where TRP is activated downstream of G-protein-coupled receptors (GPCRs), G proteins, phospholipase A2, or unknown mechanoreceptors (Kahn-Kirby et al., 2004; Hardie and Franze, 2012; Hill-Eubanks et al., 2014; Plant, 2014). In L. polyedra, GPCRs or direct activation of G proteins are likely primary receptor candidates, because mechanically stimulated luminescence is inhibited by the G-protein inhibitor GDPPS (Chen et al., 2007).

In summary, evidence for TRP channels in the eukaryotic protist L. polyedra and a role for them in mechanosensing emphasize the evolutionary conservation of both the channel structures and their function. Our results suggest a mechanosensing signaling pathway where TRP is activated by PLC, a mechanism that is found in TRP-mediated signaling pathways from C. elegans to Drosophila and mammals. The finding of TRP-like channels in the alveolate protist L. polyedra supports the presence of TRP channels and their role in mechanosensing in ancestors of the Unikonta and Bikonta.

Acknowledgments

We thank L. Gerwick for advice on bioinformatics, M. Topel for advice on phylogenetics, E. Selander for advice on statistics, and M. Beekman and J. Stires for helpful discussions. JBL was supported by the Carl Trygger Foundation for Scientific Research (CTS 14:294 and CTS KF16:11). This study was supported in part by the UC San Diego Academic Senate.

Data Accessibility

The Lingulodinium polyedra transcriptome generated in this study, L. polyedra TRP-like polypeptide sequences, and a video showing L. polyedra luminescence in response to capsaicin are available from the Zenodo open research repository (see Lindstrom et al., 2017). The associated online supplementary material contains methods for assembly and annotation of the L. polyedra transcriptome (Appendix S1, available online), a list of TRP sequences used in phylogenetic inference (Table A1), and original data from the Hidden Markov Model sequence prediction of L. polyedra TRP-like polypeptides (Fig.A1).

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J. B. LINDSTROM (*), N. T. PIERCE, AND M. I. LATZ ([dagger])

Scripps Institution of Oceanography, University of California, San Diego, 9500 Oilman Drive, La Jolla, California 92093

Received 11 April 2017; Accepted 15 June 2017; Published online 3 January 2018.

(*) Present address: Department of Biological and Environmental Sciences, University of Gothenburg, Box 100, S-405 30 Gothenburg, Sweden.

([dagger]) To whom correspondence should be addressed. E-mail: mlatz@ucsd.edu. Abbreviations: AA, amino acids; AMTB hydrochloride, N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride; Ce, Caenorhabditis elegans; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster; DMSO, dimethyl sulfoxide; Dr, Danio rerio; FSW, filtered sea-water; [Gd.sup.3+], gadolinium; GsMTx4, Grammostola spatulata mechanotoxin 4; HC067047, 2-Methyl-l-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-lH-pyrrole-3-carboxamide; HMM, Hidden Markov Model: Hs, Homo sapiens; Lp, Lingulodinium polyedra; Mb, Monosiga brevicollis; ML204, 4-Methyl-2-(l-piperidinyl)-quinoline; ORF, open reading frame; [PIP.sub.2], Phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; Pt, Paramecium tetraurelia; RHC80267, O, O'-[l,6-Hexanediylbis,(iminocarbonyl)ldioxime cyclohexanone; RN1734, 2,4-Dichloro-N-isopropyl-N-(2-isopropylaminoethyl) benzenesulfonamide; RN 1747, 1-(4-Chloro-2-nitrophenyl)sulfonyl-4-benzylpiperazine; TMHMM, transmembrane helix prediction; TRP, transient receptor potential channel; U73122, l-[6-[((17P)-3-Methoxyestra-l,3,5[10]-trien-17-yl)amino]hexyl]-1 H-pyrrole-2,5-dione.

Online enhancements: video, supplemental appendix.

Appendix A
Table A1
Transient receptor potential (TRP) channel amino acid sequences used
with Lingulodinium polyedra TRP-like sequences in phylogenetic
inference

Species                    Sequence in phylogenetic tree

Caenorhabditis elegans     Ce_OSM9 (ACQ44034)
Chlamydomonas reinhardtii  Cr_TRPl (XP_001698345.1)
                           Cr_TRP11 (BAK18929)
Drosophila melanogaster    Dm_TRP (AAA28976)
                           Dm_TRPA (NP_648263.5)
                           Dm_TRPgamma (NP_609802.1)
                           Dm_TRPL (NP_476895.1)
                           Dm_TRPM (NP_001036548.1)
                           Dm_TRPML (NP_649145.1)
                           Dm_TRPN (NP_523483.2)
                           Dm_TRPP (NP_609561.2)
                           Dm_TRPV (AAP57097.1)
Danio rerio                Dr_TRPA2 (NP_001007067.1)
                           Dr_TRPC2 (NP_001025337.1)
                           Dr_TRPM6 (ADT91627)
                           Dr_TRPML1 (NP_001315094.1)
                           Dr_TRPN1 (NP_899192.1)
                           Dr_TRPP2 (NP_001002310.1)
                           Dr_TRPV4 (NP_001036195.1)
Homo sapiens               Hs_TRPA1 (NP_015628.2)
                           Hs_TRPC1 (NP_00003295.1)
                           Hs_TRPC3 (NP_0011241701)
                           Hs_TRPC4 (NP_057263.1)
                           Hs_TRPC5 (NP_036603.1)
                           Hs_TRP6 (NP_004612.2)
                           Hs_TRP7 (NP_065122.1)
                           Hs_TRPM1 (NP_002411.3)
                           Hs_TRPM2 (NP_003298.1)
                           Hs_TRP3 (NP_066003.3)
                           Hs_TRPM4 (NP_780339.2)
                           Hs_TRPM5 (NP_055370.1)
                           Hs_TRPM6 (NP_060132.3)
                           Hs_TRPM7 (NP_060142.3)
                           Hs_TRPM8 (NP_076985.4)
                           Hs_TRPML1 (NP_065394.1)
                           Hs_TRPML2 (NP_694991.2)
                           Hs_TRPML3 (NP_060768.8)
                           Hs_TRPP1 (NP_000288.1)
                           Hs_TRPP2 (NP_057196.2)
                           Hs_TRPP3 (NP_058623.2)
Paramecium tetraurelia     Pt_TRP1 (XP_001452370.1)
                           Pt_TRP2 (XP_001444531.1)
                           PLTRP3 (XP_001429751.1)
Monosiga brevicollis       Mb_TRPAl (Monbrl | 22692 |TRPA1-1)
                           Mb_TRPC (Monbrl | 27307 |TRPC)
                           Mb_TRPM1 (Monbrl | 27446 | TRPM1)
                           Mb_TRPM2 (Monbrl | 29245 | TRPM2)
                           Mb_TRPM3 (Monbrl | 34057 | TRPM3)
                           Mb_TRPML (Monbrl | 5126 | TRPML)
                           Mb_TRPV (Monbrl | 22556 | TRPV)

Sequences were collected from http://www.jgi.doe.gov (Mb sequences)
and http://www.ncbi.nlm.nih.gov/protein (all other sequences).

Table 1
Pharmacological compounds considered for their effect on Lingulodinium
polyedra luminescence


Compound          Reported effects in TRP-mediated and mechanosensing
                  signaling pathways

Agonists
Arachidonic acid  Activates mammalian members of TRPV, TRPA, and TRPM
                  (10-150 [micro]mol [1.sup.-1]) and Drosophila TRP
                  and TRPL (2-20 [micro]mol [1.sup.-1]). Inhibition of
                  TRPA and TRPM8 is also reported (Chyb et al., 1999;
                  Oike et al., 2006; Aires et al., 2007; Andersson et
                  al., 2007; Meves, 2008; Vriens et al., 2009)
Capsaicin         Activates mammalian TRPV1 (0.1-10 [micro]mol
                  [1.sup.-1]). Stimulates TRP-related responses in
                  leach
                  (100 [micro]mol [1.sup.-1]) and mealworm (0.1
                  [micro]mol [1.sup.-1]) (Caterina et al., 1997;
                  Olszewska and Tegowska,
                  2011; Cao et al., 2013; Summers et al., 2014; Su et
                  al., 2017)
RN1747            Activates mammalian TRPV4 (1-4 [micro]mol [1.sup.-1])
                  (Vincent et al., 2009; Vriens et al., 2009; Vincent
                  and
                  Duncton, 2011)
Antagonists
Carvacrol         Inhibits mammalian TRPM7 and Drosophila TRPL (50-500
                  [micro]mol [1.sup.-1]). Activation of TRPV3 and
                  TRPA1 is also reported (Parnas et al., 2009; Chen et
                  al., 2015)
Gadolinium        Inhibits mechanosensing ion channels in eukaryotes
                  and bacteria (1 [micro]mol [1.sup.-1] -2 mmol
                  [1.sup.-1]). Inhibition of
                  voltage-gated [Ca.sup.2+] channels and activation of
                  TRPV 1 have also been reported (Yang and Sachs,
                  1989; Berrier et al., 1992; Bevan et al., 1992;
                  Lacampagne et al., 1994; Tousova et al., 2005;
                  Vriens et al., 2009; Ermakov et al., 2010; Matthews
                  et al., 2010; Bouron et al., 2015)
ML204             Inhibits mammalian TRPC4 and 5 (1-30 [micro]mol
                  [1.sup.-1]) (Miller et al., 2011; Westlund et al.,
                  2014;
                  Wei et al., 2015)
RHC80267          Inhibits breakdown of DAG to monoacylglycerol and
                  fatty acids (e.g., arachidonic acid) by inhibition
                  of DAG-lipase (30-50 [micro]mol [1.sup.-l]) (Migas
                  and Severson, 1996; Hofmann et al., 1999; Imai et
                  al.,
                  2012; Zhang et al., 2015a)
U73122            Inhibition of PLC activation (1-50 [micro]mol
                  [1.sup.-1]) (Lukacs et al., 2007; Bagher et al.,
                  2012; Klasen et al.,
                  2012; Balla, 2013)

                          Concentrations in
Compound
                  this study ([micro]mol [1.sup.-1])

Agonists
Arachidonic acid
                               5-10 (a)


Capsaicin

                              10-30 (a)


RN1747

                               1-10 (a)

Antagonists
Carvacrol
                             500


Gadolinium

                               5 (b)



ML204

                               1 (c)

RHC80267
                              10 (a)


U73122

                               1 (c)


Summary of previously published transient receptor potential
(TRP)-related effects of the pharmacological compounds used in this
study. Effects in organisms are mentioned with the main reported
effect listed first. The range of concentrations given is based on
those used in the cited studies. PLC, phospholipase C.
(a) Higher concentrations dissolve poorly in filtered seawater.
(b) Higher concentrations had inconclusive effects on luminescence and
were toxic in some experiments.
(c) Toxic in higher concentrations.

Table 2
Results of BLASTP analysis

BLASTP query sequence    Lingulodinium polyedra ORF      e-value

Hs TRPM2 (NP_003298.1)              Lp1              2.54 [e.sup.-38]
Hs TRPM8 (NP_076985.4)              Lp2              2.45 [e.sup.-29]
Hs TRPP1 (NP_000288.1)              Lp3              2.10 [e.sup.-36]
Hs TRPP2 (NP_057196.2)              Lp4              1.24 [e.sup.-38]
Hs TRPP2 (NP_057196.2)              Lp5              5.36 [e.sup.-33]
Hs TRPML2 (NP_694991.2)             Lp6              6.45 [e.sup.-18]

BLASTP query sequence    Coverage (%)  Identical sites (%)

Hs TRPM2 (NP_003298.1)        64               24
Hs TRPM8 (NP_076985.4)        82               22
Hs TRPP1 (NP_000288.1)        54               24
Hs TRPP2 (NP_057196.2)        59               27
Hs TRPP2 (NP_057196.2)        53               25
Hs TRPML2 (NP_694991.2)       39               29

Identified transient receptor potential (TRP)-like sequences in the L.
polyedra transcriptome open reading frame (ORF) database based on
BLASTP analysis using human TRP query sequences. Human sequences
represent the mammalian TRP subfamilies. BLASTP e-values, sequence
coverage, and identical sites between analyzed sequences are
displayed. Hs, Homo sapiens; Lp, Lingulodinium polyedra.
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Date:Oct 1, 2017
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