AbstractsBiology & Animal Science

Structure-function analysis of the Vesicular glutamate transporter 1 (VGLUT1) C-terminus

by Julia-Christine Jordan

Institution: Freie Universität Berlin
Department: FB Biologie, Chemie, Pharmazie
Degree: PhD
Year: 2015
Record ID: 1102997
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000099279


Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles (SVs) with the neurotransmitter (NT) glutamate. After SV fusion and NT release, SVs are recycled to maintain a constant SV supply. In order for these SVs to participate again in neurotransmission, they must be refilled. Therefore, VGLUTs need to be efficiently and correctly targeted to SVs undergoing endocytosis. Previous studies showed that the C-terminal VGLUT1 FV motif, which is part of the dileucine-like sequence, is a sorting signal for VGLUT1 trafficking to SVs. Mutations in the FV motif show slower VGLUT1 recycling (Foss et al., 2013; Voglmaier et al., 2006), but it is unknown, whether an impaired VGLUT1 retrieval consequently alters glutamatergic neurotransmission. This issue was addressed by further studying mutant variants of this VGLUT1 C-terminal dileucine-like motif sequence. Another aim of the present study was to investigate software predicted putative VGLUT1 C-terminus serine phosphorylation sites to understand their potential role for the VGLUT1 physiology and SV cycling. Additionally, the functional consequences of an entire VGLUT1 C-terminus deletion were studied as well. Therefore, VGLUT1 C-terminal mutants were expressed by lentivirus in autaptic hippocampal VGLUT1 knockout (KO) neurons and compared to a rescue of the KO neurons with expression of the VGLUT1 wildtype (WT) protein. Parameters of release and SV recycling were assessed using whole-cell voltage clamp recordings in combination with pHluorin-based imaging. With high resolution electron microscopy (EM) further information were obtained especially about SV morphology and SV number per synapse, which might be altered according to changes in vesicular release or recycling properties. Thereby, it was found out that neurons expressing a full VGLUT1 C-terminus truncation construct (VGLUT1∆496-560), where all amino acids immediately following the last 12th transmembrane domain are deleted, are not functional. Those neurons exhibit an electrophysiological and morphological VGLUT1 KO-like phenotype apparent in significantly reduced postsynaptic responses and deformed, smaller SVs compared to WT rescued neurons indicating that the SVs of VGLUT1∆496-560 are not glutamate filled. A reason for the impairments of VGLUT1∆496-560 may be protein mislocalization to vesicles. However, no crystal structure of the VGLUT1 protein exists and it is unclear, whether this deletion may have been within or close enough to the transmembrane domain causing instability of the protein and subsequent degradation. In contrast, neurons expressing a C-terminus truncation construct that was a few amino acids shorter (VGLUT1∆504-560), including a WAEPE motif sequence, can rescue the VGLUT1 KO-like phenotype to WT rescue levels, suggesting that the main function of the transporter to fill SVs with NT is here not affected. Moreover, it could be also shown that the SV morphology is unaltered in the VGLUT1∆504-560 mutant. However, the VGLUT1 recycling of VGLUT1∆504-560 is…