First published online December 1, 2006
Journal of Experimental Biology 209, 4858-4868 (2006)
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
1 Department of Biology, University of Western Ontario, London, ON, Canada
N6A 5B7
2 Southern Crop Protection and Food Research Centre, Agriculture and
Agri-Food Canada, London, ON, Canada N5V 4T3
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Fig. 1. Metazoan monoamine transporters belonging to the SLC6 family of membrane
transporters have diagnostic polypeptide motifs. These conserved motifs helped
identify three different monoamine transporter types, iDAT, a dopamine
transporter restricted to invertebrates, and two widely distributed metazoan
transporters, MAT, a catecholamine/phenolamine monoamine transporter, and
SERT, a serotonin transporter. Motifs 1, 2 and 3 are present in all three
transporter types, whereas motif 4 is a C-terminal sequence restricted to the
MAT-type catecholamine/phenolamine transporter. Motif 1 is mainly in
transmembrane domain 1 (TMD1), motif 2 in the cytoplasmic loop (CL1) between
TMDs 2 and 3 and motif 3 is in transmembrane domain 5 (TMD5). Amino acid
residues shaded in grey are consensus residues; the aspartate residue (D) in
motif 1 and the tryptophan residue (W) (rarely tyrosine, Y) in motif 3 are not
found in related nutrient amino acid transporters (NATs). The amino acids (or
site of missing amino acid in MAT) shaded in yellow are largely restricted to
one transporter type. Amino acids shaded in blue or green are restricted to
subsets within a transporter type. Abbreviations for the other amino acids
follow the single-letter code. The positions of the primer sites used in
RT-PCR screening for partial transporter sequences are shown above the
alignment.
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Fig. 2. Comparison of partial SLC6 monoamine transporter sequences in the Metazoa.
The dendrogram combines our RT-PCR data with sequence data available online.
The major resulting clusters are as labelled. Note that chordate DATs (cDATs)
group more closely with vertebrate NETs than with invertebrate DATs (iDATs).
The NCBI nucleotide and protein sequence accession numbers and other genome
resource data used to assemble this figure are listed in supplementary
material Table S1). Most of the invertebrate sequences are included in the
NCBI PopSets #70797604, #113204555, #113204575 and #110734541. The scale bar
shows evolutionary distance in number of substitutions per site.
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Fig. 3. Sequence comparisons of complete (or near-complete) metazoan monoamine
transporter sequences obtained from BLAST searches online and a Lumbricus
terrestris OAT sequence (NCBI accession #DQ813341) from this study. The
dendrogram confirms our tentative groupings based on the presence or absence
of certain diagnostic amino acid residues in the partial transporter sequences
(Fig. 1). Note that protostome
OATs and deuterostome DATs/NETs form a single cluster (MAT). A NET-like gene
is apparently lacking from protostome genomes and an OAT-like gene from
deuterostome genomes such as echinoderms and chordates. The NCBI nucleotide
and protein accession numbers and other genome resources used are listed in
supplementary material Table S2. The scale bar shows evolutionary distance in
number of substitutions per site.
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Fig. 4. Stable expression of Lumbricus terrestris OAT transporter RNA in
transformed Sf9 insect cells. The relative affinity of LutOAT for
neurotransmitter monoamines (MA) was determined by exposing transformed Sf9
cells to radiolabelled isotopes of the phenolamines tyramine (TA) and
octopamine (OA), and the catecholamines dopamine (DA) and norepinephrine (NE)
in vitro. Cells expressing LutOAT take up all four monoamines with
high-affinity and in a Na+-dependent manner. TA and OA uptake
saturates at mid-nanomolar substrate concentrations, whereas DA and NE uptake
saturates at lowmicromolar concentrations. Uptake under non-saturating
substrate concentrations is log-linear (as indicated by the slope of the
broken lines). There is an inverse relationship between affinity and transport
capacity. LutOAT has the highest affinity and the lowest transport capacity
for TA; the opposite holds for NE. The estimated kinetic values are given in
Table 1. Each point represents
the mean ± s.d., N=3-5 independent trials.
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Fig. 5. Suggested origins of the three monoamine transporter types in the
Bilateria. Homologues of the serotonin transporter gene, SERT, are expressed
in all phyla examined to date. Although presumed to be ancestral, the
invertebrate DAT gene (iDAT) may have been lost in some invertebrate phyla
(Annelida, possibly Mollusca) and in the Chordata. The ancestral
catecholamine/phenolamine transporter (MAT) persists today as invertebrate OAT
and the chordate paralogues NET and cDAT. The chordate NET and cDAT genes
appear to have arisen through duplication of a MAT gene following the loss of
an invertebrate-type DAT gene in a basal protochordate. The loss, or absence
of expression, of some monoamine transporter genes in different phyla (iDAT in
Annelida, iDAT and MAT in Mollusca) or in sub-phyletic taxa (DAT in
Urochordata, OAT in some Insecta, SERT in Hymenoptera) appears to be a
commonly repeated theme in the Metazoa.
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© The Company of Biologists Ltd 2006
