Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

57-POS Board 20 A CELL-IMPERMEABLE FLUORESCENT SENSOR FOR SELECTIVE LABELING OF GLUTAMATE IN SYNAPTIC VESICLES

Kevin D. Gillis 1,2 ; Peeyush Yadav 4 ; Ming Xu 4 ; Xin Liu 2 ; Clarissa Durie 3 ; Timothy E Glass 4 ; 1 University of Missouri, Chemical and Biomedical Engineering, Columbia, MO, USA 2 University of Missouri, Dalton Cardiovascular Research Center, Columbia, MO, USA

3 University of Missouri, Biochemistry, Columbia, MO, USA 4 University of Missouri, Chemistry, Columbia, MO, USA

Glutamate is an important excitatory neurotransmitter in the central nervous system and is stored in synaptic vesicles at a concentration of ~100 mM. There is a lack of tools for fluorescent imaging of glutamate within vesicles in living cells. Here we report a novel cell-impermeable fluorescent sensor (NS570) for the selective imaging of glutamate upon refilling of recycled vesicles. NS570 has two reactive groups: an aldehyde which binds to the amine group of glutamate and a boronic acid moiety which binds to the carboxylic acid group of glutamate. In addition, two carboxylic acid “tails” are introduced in the sensor to make it cell impermeable, so that it can only be loaded into synaptic vesicles by endocytosis during vesicle cycling. Background fluorescence of the sensor without binding is minimal, and increases ~2700-fold in vitro with a saturating concentration of glutamate (K d ~ 20 mM, absorbance maxima ~485 nm, emission maxima ~570 nm). The enhancement of fluorescence upon binding GABA is much more modest (~95-fold), presumably because it doesn’t bind to the boronic acid moiety. Experiments with cultured hippocampal neurons show minimal background fluorescence when NS570 (25 mM) is included in the bath solution (515 nm excitation). Stimulation with a high K + solution or electrical stimulation leads to fluorescence increases within neurons, presumably as the sensor is taken up by endocytosed vesicles and turned on upon refilling of vesicles with transmitter. A second round of stimulation following washout of sensor from the bath leads to fluorescence decreases, presumably due to release of neurotransmitter-bound sensor during exocytosis. Stopped-flow measurements indicate binding of glutamate to the sensor is slow (minutes), which may be insufficient to resolve the dynamics of refilling of vesicles by transmitter. Supported by NSF 2203359.

92