TY - JOUR
T1 - Characterization of a Dopamine Transporter and Its Splice Variant Reveals Novel Features of Dopaminergic Regulation in the Honey Bee
AU - Zhang, Vicky
AU - Kucharski, Robert
AU - Landers, Courtney
AU - Richards, Sashika N.
AU - Bröer, Stefan
AU - Martin, Rowena E.
AU - Maleszka, Ryszard
N1 - Copyright © 2019 Zhang, Kucharski, Landers, Richards, Bröer, Martin and Maleszka.
Funding Information:
We thank Joanna Maleszka for performing the in situ hybridization and tissue preparation, Paul Helliwell for beekeeping, Sarah Shafik for slicing and imaging samples for the oocyte immunofluorescence microscopy, and Angelica Bröer for assistance with the initial Xenopus oocyte transport assays. Funding. This work was supported by the Australian Research Council (Discovery Grant DP160103053 to RM and Future Fellowship FT160100226 to REM) and Australian Government Research Postgraduate Awards to VZ and SR.
Publisher Copyright:
© Copyright © 2019 Zhang, Kucharski, Landers, Richards, Bröer, Martin and Maleszka.
PY - 2019/11/1
Y1 - 2019/11/1
N2 - Dopamine is an important neuromodulator involved in reward-processing, movement control, motivational responses, and other aspects of behavior in most animals. In honey bees (Apis mellifera), the dopaminergic system has been implicated in an elaborate pheromonal communication network between individuals and in the differentiation of females into reproductive (queen) and sterile (worker) castes. Here we have identified and characterized a honey bee dopamine transporter (AmDAT) and a splice variant lacking exon 3 (AmDATΔex3). Both transcripts are present in the adult brain and antennae as well as at lower levels within larvae and ovaries. When expressed separately in the Xenopus oocyte system, AmDAT localizes to the oocyte surface whereas the splice variant is retained at an internal membrane. Oocytes expressing AmDAT exhibit a 12-fold increase in the uptake of [3H]dopamine relative to non-injected oocytes, whereas the AmDATΔex3-expressing oocytes show no change in [3H]dopamine transport. Electrophysiological measurements of AmDAT activity revealed it to be a high-affinity, low-capacity transporter of dopamine. The transporter also recognizes noradrenaline as a major substrate and tyramine as a minor substrate, but does not transport octopamine, L-Dopa, or serotonin. Dopamine transport via AmDAT is inhibited by cocaine in a reversible manner, but is unaffected by octopamine. Co-expression of AmDAT and AmDATΔex3 in oocytes results in a substantial reduction in AmDAT-mediated transport, which was also detected as a significant decrease in the level of AmDAT protein. This down-regulatory effect is not attributable to competition with AmDATΔex3 for ER ribosomes, nor to a general inhibition of the oocyte’s translational machinery. In vivo, the expression of both transcripts shows a high level of inter-individual variability. Gene-focused, ultra-deep amplicon sequencing detected methylation of the amdat locus at ten 5′-C-phosphate-G-3′ dinucleotides (CpGs), but only in 5–10% of all reads in whole brains or antennae. These observations, together with the localization of the amdat transcript to a few clusters of dopaminergic neurons, imply that amdat methylation is positively linked to its transcription. Our findings suggest that multiple cellular mechanisms, including gene splicing and epigenomic communication systems, may be adopted to increase the potential of a conserved gene to contribute to lineage-specific behavioral outcomes.
AB - Dopamine is an important neuromodulator involved in reward-processing, movement control, motivational responses, and other aspects of behavior in most animals. In honey bees (Apis mellifera), the dopaminergic system has been implicated in an elaborate pheromonal communication network between individuals and in the differentiation of females into reproductive (queen) and sterile (worker) castes. Here we have identified and characterized a honey bee dopamine transporter (AmDAT) and a splice variant lacking exon 3 (AmDATΔex3). Both transcripts are present in the adult brain and antennae as well as at lower levels within larvae and ovaries. When expressed separately in the Xenopus oocyte system, AmDAT localizes to the oocyte surface whereas the splice variant is retained at an internal membrane. Oocytes expressing AmDAT exhibit a 12-fold increase in the uptake of [3H]dopamine relative to non-injected oocytes, whereas the AmDATΔex3-expressing oocytes show no change in [3H]dopamine transport. Electrophysiological measurements of AmDAT activity revealed it to be a high-affinity, low-capacity transporter of dopamine. The transporter also recognizes noradrenaline as a major substrate and tyramine as a minor substrate, but does not transport octopamine, L-Dopa, or serotonin. Dopamine transport via AmDAT is inhibited by cocaine in a reversible manner, but is unaffected by octopamine. Co-expression of AmDAT and AmDATΔex3 in oocytes results in a substantial reduction in AmDAT-mediated transport, which was also detected as a significant decrease in the level of AmDAT protein. This down-regulatory effect is not attributable to competition with AmDATΔex3 for ER ribosomes, nor to a general inhibition of the oocyte’s translational machinery. In vivo, the expression of both transcripts shows a high level of inter-individual variability. Gene-focused, ultra-deep amplicon sequencing detected methylation of the amdat locus at ten 5′-C-phosphate-G-3′ dinucleotides (CpGs), but only in 5–10% of all reads in whole brains or antennae. These observations, together with the localization of the amdat transcript to a few clusters of dopaminergic neurons, imply that amdat methylation is positively linked to its transcription. Our findings suggest that multiple cellular mechanisms, including gene splicing and epigenomic communication systems, may be adopted to increase the potential of a conserved gene to contribute to lineage-specific behavioral outcomes.
KW - biogenic amine, dopaminergic neurons, social behavior, heteromeric protein, spliced transporter, DNA methylation
KW - spliced transporter
KW - social behavior
KW - dopaminergic neurons
KW - DNA methylation
KW - biogenic amine
KW - heteromeric protein
UR - http://www.scopus.com/inward/record.url?scp=85075374983&partnerID=8YFLogxK
UR - https://www.frontiersin.org/articles/10.3389/fphys.2019.01375?utm_source=researcher_app&utm_medium=referral&utm_campaign=RESR_MRKT_Researcher_inbound
UR - http://www.mendeley.com/research/characterization-dopamine-transporter-splice-variant-reveals-novel-features-dopaminergic-regulation
U2 - 10.3389/fphys.2019.01375
DO - 10.3389/fphys.2019.01375
M3 - Article
C2 - 31736791
SN - 1664-042X
VL - 10
SP - 1
EP - 16
JO - Frontiers in Physiology
JF - Frontiers in Physiology
M1 - 1375
ER -