Ancestry of Neuronal Monoamine Transporters in the Metazoa
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4858 The Journal of Experimental Biology 209, 4858-4868 Published by The Company of Biologists 2006 doi:10.1242/jeb.02607 Ancestry of neuronal monoamine transporters in the Metazoa Stanley Caveney1,*, Wendy Cladman1, LouAnn Verellen2 and Cam Donly2 1Department of Biology, University of Western Ontario, London, ON, Canada N6A 5B7 and 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, Canada N5V 4T3 *Author for correspondence (e-mail: [email protected]) Accepted 18 October 2006 Summary Selective Na+-dependent re-uptake of biogenic SERT currently found throughout bilaterian phyla, a DAT monoamines at mammalian nerve synapses is now restricted in distribution to protostome invertebrates accomplished by three types of solute-linked carrier family and echinoderms and a third monoamine transporter 6 (SLC6) membrane transporter with high affinity for (MAT), widely represented in contemporary Bilateria, that serotonin (SERTs), dopamine (DATs) and norepinephrine is selective for catecholamines and/or phenolamines. (NETs). An additional SLC6 monoamine transporter Chordate DATs, NETs, epinephrine transporters (ETs) (OAT), is responsible for the selective uptake of the and arthropod and annelid OATs all belong to the MAT phenolamines octopamine and tyramine by insect clade. Contemporary invertebrate and chordate DATs neurons. We have characterized a similar high-affinity belong to different SLC6 clades. Furthermore, the genes phenoloamine transporter expressed in the CNS of the for dopamine and norepinephrine transporters of earthworm Lumbricus terrestris. Phylogenetic analysis of vertebrates are paralogous, apparently having arisen its protein sequence clusters it with both arthropod through duplication of an invertebrate MAT gene after phenolamine and chordate catecholamine transporters. To the loss of an invertebrate-type DAT gene in a basal clarify the relationships among metazoan monoamine protochordate. transporters we identified representatives in the major branches of metazoan evolution by polymerase chain Supplementary material available online at reaction (PCR)-amplifying conserved cDNA fragments http://jeb.biologists.org/cgi/content/full/209/24/4858/DC1 from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence Key words: Bilateria, protostome, deuterostome, monoamine, data suggest that the presumed common ancestor of neurotransmitters, transporter, SLC6 family, octopamine, tyramine, modern-day Bilateria expressed at least three functionally dopamine, norepinephrine, serotonin, earthworm, Lumbricus distinct monoamine transporters in its nervous system: a terrestris Introduction developmental characteristics (Phillipe et al., 2005). Analysis The molecular chemistry of the metazoan nervous system of genomic and transcriptome sequences from different has a common evolutionary heritage. Serotonergic and bilaterian groups suggests that Urbilateria possessed a full dopaminergic neurons date back at least 600 million years to complement of genes coding for neuronal proteins involved in the stem metazoan, an ancient crawling worm proposed to be the synthesis, reception and selective re-uptake of monoamine the common ancestor of the Cnidaria (radially symmetric neurotransmitters such as serotonin and dopamine (DA). Two organisms such as jellyfish, sea anemone and hydra) and the other monoaminergic pathways have a restricted distribution in Bilateria (bilaterally symmetric animals that comprise most the bilaterian nervous system. Neuronal pathways based on present-day animals) (Hay-Schmidt, 2000; Anctil et al., 2002; the phenolamines tyramine (TA) and octopamine (OA) are Bouchard et al., 2003; Raible and Arendt, 2004). In turn, all characteristic of protostomes (Pflüger and Stevenson, 2005; Bilateria are thought to share a common ancestor named Evans and Maquiera, 2005), whereas those based on the Urbilateria (De Robertis and Sasai, 1996; Raible and Arendt, catecholamines norepinephrine (NE) and epinephrine (E) are 2004). Modern bilaterian animals fall into either the characteristic of deuterostomes (Vincent et al., 1998). Protostomia (major phyla Annelida, Mollusca, Arthropoda, The neuronal expression of plasma membrane transporters Nematoda and Platyhelmintha) or the Deuterostomia (phyla involved in neurotransmitter re-uptake is a diagnostic Echinodermata and Chordata), based on molecular genetic and component of monoaminergic systems in the Bilateria (Torres THE JOURNAL OF EXPERIMENTAL BIOLOGY Ancestry of neuronal monoamine transporters 4859 et al., 2003). The fully sequenced genomes of animals degenerate primers and 2.5·U Taq DNA Polymerase representative of major bilaterian phyla, such as those of the (Invitrogen). The PCR conditions were 94°C for 2·min, followed flatworm, fruitfly, nematode and human, all contain serotonin by 35 cycles of 94°C for 30·s, 55°C for 30·s and 72°C for 1·min, transporters (SERTs) and dopamine transporters (DATs) followed by one 5-min hold at 72°C. (Pörzgen et al., 2001; Caveney and Donly, 2002). Other Products from PCR were separated on agarose gels and monoamine transporters expressed by monoaminergic neurons bands estimated to be of the appropriate size were ligated into include an OA/TA transporter (OAT) associated with arthropod pGEM-T Easy (Promega, Madison, WI, USA) and transformed octopaminergic pathways (Malutan et al., 2002; Donly and into XL1-blue cells (Stratagene, La Jolla, CA, USA). Plasmids Caveney, 2005) and the norepinephrine/epinephrine transporter were subjected to dideoxynucleotide chain-termination (NET) associated with chordate adrenergic pathways sequencing using an ABI Prism BigDye Terminator Cycle (Apparsundaram et al., 1997; Roubert et al., 2001). These Sequencing Kit and resolved with an ABI 3100 DNA monoamine transporters are all metazoan members of the sequencer (Applied Biosystems, Foster City, CA, USA). solute-linked carrier (SLC) family 6 [or alternatively sodium:neurotransmitter symporter (SNF) family] of Na+- Cloning and expression of a Lumbricus MAT dependent nutrient transporters that are expressed in both Using the sequence of the internal DAT0/DAT3 fragment for prokaryotes (Androutsellis-Theotokis et al., 2003; Yamashita et Lumbricus terrestris, nested pairs of primers were synthesized al., 2005) and eukaryotes (Chen et al., 2004; Boudko et al., for 5Ј and 3Ј rapid amplification of cDNA ends-PCR (RACE- 2005b). Monoamine transporters are highly conserved and PCR). RACE-PCR was performed using the FirstChoice RLM widely expressed in the bilaterian nervous system. Their protein RACE kit (Ambion) as directed by the supplier. From the fully structure and substrate kinetics may provide information on the assembled sequence, primers were synthesized to amplify the origin and number of genes that encode SLC6 monoamine complete open reading frame (ORF) (Lut8R: 5Ј-CGGCA- transporters in the Metazoa (Chen et al., 2004; Höglund et al., GCTCTCGAAGGATACCAT-3Ј and Lut9F: 5Ј-GAAGCA- 2005). Do the genes encoding the transporter for each specific GAGACCGAGTGACGAGGA-3Ј). The resulting 2.4·kb monoamine in the metazoan CNS derive from a common product was amplified using EasyA polymerase (Stratagene) ancestor, and if not, are there differences in the gene and cloned in pGEM-T Easy. Three independently amplified complements present in the different bilaterian lineages? What products were sequenced to avoid polymerase errors. was the ancestral monoamine transported by the different types The cloned insert was transferred to the pIZ/V5-His vector of transporter? And what is the relationship between protostome for expression using the InsectSelect System (Invitrogen). OA and deuterostome NE transporters? Purified plasmid (6·g) was used to transfect Sf9 cells in To address these questions, we have used both existing and 60·mm culture plates using Cellfectin Reagent as instructed by new structural and functional data on SLC6 monoamine the supplier (Invitrogen). Stably transformed lines derived from transporters representing many metazoan phyla. We have also individual dishes were established and maintained in the cloned and characterized a phenolamine transporter from the presence of 100·g·ml–1 Zeocin. CNS of the earthworm. The results suggest that OA and NE are ancient neurotransmitters in the protostome and Transport assays deuterostome lineages, respectively, and that their transporters Transport assays were performed in 12-well plates (Falcon; share a common origin. Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described (Gallant et al., 2003). Sf9 cells stably expressing LutOAT were seeded at a density of 800·000 per well in 1·ml Materials and methods of SF-900 II SFM medium (Invitrogen) and used 24·h later. The Reverse transcription-polymerase chain reaction (RT–PCR) wells were briefly rinsed and then incubated for an hour in a + –1 –1 Template for amplification was obtained from each organism Na -containing saline (11.2·mmol·l MgCl2, 11.2·mmol·l –1 –1 by dissecting the brain and/or nerve cord directly into RNAlater MgSO4, 53.5·mmol·l NaCl, 7.3·mmol·l NaH2PO4, (Ambion, Austin, TX, USA). Total RNA was isolated using 55·mmol·l–1 KCl and 76.8·mmol·l–1 sucrose at pH 7). The cells Trizol reagent (Invitrogen, Burlington, ON, Canada) and made were then exposed to 500·l Na+ saline containing either –1 –1 3 up in DEPC-treated H2O. Synthesis of cDNA was performed 29·nmol·l to 0.29·mol·l H-DA (specific activity 34.8 using 10·U Superscript II reverse transcriptase (Invitrogen) and Ci·mmol·l–1;