Abstract
Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins. VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A-E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light-dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1.