Abstract
Whilst the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles, accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. In this study, we extend the role of the vesicular acetylcholine transporter (VAChT) to include regulating the transmitter content of synaptic vesicles. We report that manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, we find that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic mEPSCs recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked synaptic vesicle release. Finally, a complete deletion of the PolyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure synaptic vesicles are filled to physiologically-appropriate levels remain unknown. Our study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission.
Significance Statement Neurotransmitter-loading of synaptic vesicles is tightly regulated and underpins the quantal theory of neurotransmission. However, although observed at every synapse studied, the mechanistic basis that ensures vesicle-filling stops at a fixed, pre-determined, level remains poorly understood. In this study we identify a C-terminal poly-glutamine region in the Drosophila VAChT to be critical for vesicle-loading of ACh. Reduction or extension of this region, by just one glutamine residue, is sufficient to increase or decrease, respectively, the amount of ACh loaded. Our work significantly advances the field of synaptic physiology by identifying a region of a vesicular transporter that regulates the extent to which synaptic vesicles are filled.
Footnotes
Conflict of Interest Statement: SWV and JG received financial support from Syngenta Ltd. Syngenta also contributed to study design and the preparation of the manuscript, but did not have any role in data collection, data analysis or decision to publish. RAB declares no conflict of interests.
Biotechnology and Biological Sciences Research Council (BBSRC) [BB/L027690/1]; Wellcome Trust (Wellcome) [087742/Z/08/Z]
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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