Characterization of a Dopamine Transporter and Its Splice Variant Reveals Novel Features of Dopaminergic Regulation in the Honey Bee

Abstract

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.

Exploring DNA Methylation Diversity in the Honey Bee Brain by Ultra-Deep Amplicon Sequencing

“…It is conceivable that in our dataset such a limited de novo methylation is represented by the infrequent patterns. This notion is supported by our recent finding that the full extent of CpG methylation in a gene encoding AmDAT (dopamine transporter) could only be revealed by ultra-deep sequencing [ 26 ]. Given the restricted expression of AmDAT in just four clusters of neurons in the honey bee brain [ 27 ], this result is not surprising, and in addition, it reinforces the idea that at least in some cases DNA methylation is positively correlated with transcription [ 25 ].…”

Robustness of the honeybee neuro-muscular octopaminergic system in the face of cold stress

“…Especially the latter would probably be an important mechanism to deal efficiently with limited resources under extreme conditions ( Schroeder and Jordan, 2012 ) and simultaneously maintain flight muscle functionality. Future studies could address these issues by combining different methods, which may include electrochemical microsensors ( Phillips and Wightman, 2003 ; Jarriault et al, 2018 ), electrophysiological recordings ( Ting and Phillips, 2007 ) and molecular and functional analyses of honeybee monoamine transporters ( Torres et al, 2003 ; Zhang et al, 2019 ).…”

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT

“…34578), quantified using the ImageJ software [ 112 ], and expressed as a percentage of the band intensity measured for PfMDR1 NYSND . Total protein staining was used to evaluate sample loading and efficiency of transfer [ 7 , 113 ]. Briefly, the nitrocellulose membranes were stained with Ponceau S for 5 minutes, then de-stained using ultrapure water.…”

“…The band was not present in ne. Oocytes expressing a HA-tagged version of the Apis mellifera dopamine transporter (HA EL2 -AmDAT) at the plasma membrane serve as a positive control [ 113 ]. The length of the scale bar is 50 μm.…”