Bouchard C et al. (2019), A SLC6 transporter cloned from the lion's mane ...

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PLoS One 2019 Jun 19;146:e0218806. doi: 10.1371/journal.pone.0218806.

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A SLC6 transporter cloned from the lion's mane jellyfish (Cnidaria, Scyphozoa) is expressed in neurons.

Bouchard C , Boudko DY , Jiang RHY .

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In the course of recent comparative genomic studies conducted on nervous systems across the phylogeny, current thinking is leaning in favor of more heterogeneity among nervous systems than what was initially expected. The isolation and characterization of molecular components that constitute the cnidarian neuron is not only of interest to the physiologist but also, on a larger scale, to those who study the evolution of nervous systems. Understanding the function of those ancient neurons involves the identification of neurotransmitters and their precursors, the description of nutrients used by neurons for metabolic purposes and the identification of integral membrane proteins that bind to those compounds. Using a molecular cloning strategy targeting membrane proteins that are known to be present in all forms of life, we isolated a member of the solute carrier family 6 from the scyphozoan jellyfish Cyanea capillata. The phylogenetic analysis suggested that the new transporter sequence belongs to an ancestral group of the nutrient amino acid transporter subfamily and is part of a cluster of cnidarian sequences which may translocate the same substrate. We found that the jellyfish transporter is expressed in neurons of the motor nerve net of the animal. To this end, we established an in situ hybridization protocol for the tissues of C. capillata and developed a specific antibody to the jellyfish transporter. Finally, we showed that the gene that codes for the jellyfish transporter also expresses a long non-coding RNA. We hope that this research will contribute to studies that seek to understand what constitutes a neuron in species that belong to an ancient phylum.

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	Fig 1. Evolutionary relationships of the newly identified CcSLC6_NAT1 protein with 78 selected SLC6 transporters.The tree shows the major subfamilies of the SLC6 family: The NeuroTransmitter-specific Transporters (NTT) subfamilies and the Nutrient Amino acid (NAT) subfamily. The C. capillata SLC6 transporter belongs to the NAT subfamily and forms a cluster with related taxa including Nematostella representing the closest homologs of the cloned jellyfish transporter. The cnidarian transporters cluster together with characterized B0 and IMINO transporters of the NAT subfamily. Other shaded clusters indicate GABAT (GABA transporters), DAT (dopamine transporters) and SERT (serotonin transporters) of the NTT subfamily. Species abbreviation included in leave strings are: BACSU: Bacillus subtilis (strain 168); BIVA: Bathymodiolus septemdierum; BRAFL: Branchiostoma floridae; CAEEL: Caenorhabditis elegans; CAPTE: Capitella teleta; CIOIN: Ciona intestinalis; CIOSA: Ciona savignyi; CRAGI: Crassostrea gigas; DROME: Drosophila melanogaster; HAEIN: Haemophilus influenzae (ATCC 51907); HUMAN: Homo sapiens; HYDVU: Hydra vulgaris; LINUN: Lingula unguis; LOTGI: Lottia gigantea; METJA: Methanocaldococcus jannaschii (ATCC 43067); MIZYE: Mizuhopecten yessoensis; NEMVE: Nematostella vectensis; STYPI: Stylophora pistillata; ORBIC: Orbicella faveolata; TRIAD: Trichoplax adhaerens; 9METZ: Trichoplax sp. H2. The transporters from prokaryotes were used as an outgroup in the tree. Numbers shown next to the nodes are local support values represented as proportions. UniProt IDs of protein sequences used to assemble this figure are indicated at the ends of branches. Scale bar represents the number of substitutions per site.
	Fig 2. Sodium and chloride binding sites as identified in the bacterial AaLeuT and the human serotonin (SERT) transporters.Transmembrane domains (TM I, II, VI, VII and VIII) that depict residues that interact with sodium 1 and 2 as well as with chloride are highlighted with the background colors blue for Na1 sites, orange for Na2 sites and green for Cl- sites. Numbers located at the top of the alignment correspond to residue numbers of AaLeuT known to interact with sodium 1 and 2, while residues numbered at the bottom of the alignment are those known to interact with chloride in SERT. The sites that coordinate the binding of sodium in the jellyfish sequence are strictly conserved for Na1 while only partly conserved for Na2. Although CcSLC6 is likely to translocate its substrate in presence of sodium, the fact that position 309 in the jellyfish sequence is a threonine as it is in AaLeuT may suggest that CcSLC6 may not rely absolutely on Cl- (see Results section for more details). The alignment is constituted of the CcSLC6_NAT1 jellyfish sequence (Accession: MH737701 / protein ID: QBP15011) along with the following three sequences for which a crystalized form has been determined: A. aeolicus AaLeuT (PDB ID 2A65), human serotonin transporter SERT Hum (NP_001036.1) and dopamine Drosophila melanogaster transporter DopaT Dm (NP_523763). The alignment also includes characterized human (Hum) as well as cnidarian sequences which are homologues of the CcSLC6_NAT1 sequence in the phylogenetic tree (Fig 1). They are identified as follows: S6A15 Hum (NP_877499HS), S6A17 Hum (NP_001010898), S6A18 Hum (NP_872438.2), S6A19 Hum (NP_001003841), S6A20 Hum (NP_064593), A7RFF4 NEMVE for Nematostella vectensis (XP_001641770.1), ORBIC for Orbicella faveolata (XP_020611607), and T2M528 HYDVU for Hydra vulgaris (XP_012559838). The sequences were aligned using ClustalW tool in MacVector (v. 15.5.4).
	Fig 3. Distribution of the CcSLC6-NAT1 transcripts in the MNN neurons of the peri-rhopalial tissue.in situ hybridization experiments were performed on whole-mount peri-rhopalial tissue using digoxigenin-labelled cRNA probes transcribed from the full-length cDNA of CcSLC6_NAT1. (A) Epithelial cell-free preparations show two neurons (left and right panels) of the MNN attached to a transparent mesoglea. The purple precipitate in the cells highlights zones where the transporter transcripts are expressed. The transcripts are predominantly present in the cytosol of the neuronal somata, in the periphery of the nuclei (n), and occasional labeling is also observed in the processes (red arrowhead in left panel). Two arrows in right panel of (A) shows the process of one neuron that forms an axosomatic contact with the labelled neuron. (B) An in situ hybridization experiment conducted on intact peri-rhopalial tissue. Labelled somata of the neurons are seen through the transparent epithelial layer. No labelling was observed in epithelial cells. (C) The labeling obtained in the control experiment using the sense probe suggests that the transporter gene may undergo bidirectional transcription. Double arrowheads indicate two processes that emerged from one neuronal soma, while a third process lay at the opposite pole of the soma, typical of tripolar nerve cells.

	Fig 5. Cellular localization of the CcSLC6_NAT1 protein in the peri-rhopalial tissue.Immunohistochemistry conducted on intact peri-rhopalial tissue using the polyclonal CcSLC6_NAT1 antibodies (green). The MNN neurons expressed the CcSLC6_NAT1 transporter robustly on the surface of the cytoplasmic membrane.
	Fig 6. Heterologous expression of the CcSLC6_NAT1 protein showed that the antibody recognizes the C. capillata transporter.The expression vector contained the GFP gene under the control of a promoter independent from the transporter’s promoter. (A) All cells that were transfected with the recombinant vector expressed GFP (green) in the cytosol of the HEK-293 cells. (B) GFP-expressing cells also expressed the transporter as revealed by the immunofluorescence experiment using the CcSLC6_NAT1 antibody (red). (C) Merged images (A) and (B): all cells expressing GFP (green) also expressed the CcSLC6_NAT1 transporter (red). Expression of the transporter was targeted at the plasma membrane (arrowheads). (D) Control (merged image). Immunocytochemistry experiment was also performed on transfected cells using the serum of the rabbit prior to immunization. The rabbit pre-serum did not label the transfected cells (absence of red fluorescence).

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