The genetically encoded fluorescent glutamate sensor, iGluSnFR3, characterized by a high dynamic range and rapid on -kinetics, is an attractive sensor for glutamate imaging in the central nervous system. However, kinetic variants with ultrafast or slow off -kinetics are needed to broaden the spectrum of applications from monitoring rapid synaptic glutamate transients to mesoscale imaging of brain activity. Here, we report binding-site variant S72T (iGlu3Fast) optimized for reporting fast glutamate release at individual sites with a 57-fold fluorescence enhancement and 2 ms decay t 1/2 in solution, ∼5-fold faster than iGluSnFR3. In contrast, variants D25A (iGlu3Slow1) and D25R (iGlu3Slow2) displayed slow off -kinetics (decay t 1/2 43 and 30 ms, respectively, 20 o C), while retaining high dynamic ranges (48 and 65, respectively). Glutamate concentration dependence of the on -kinetics of iGluSnFR3 variants fit to a hyperbolic function that describes a two-step reaction in which rapid binding is followed by a conformational change evoking fluorescence enhancement. The rate limiting on -rate was 832 ± 148 s −1 and 1736 ± 278 s -1 , for iGlu3fast and iGlu3Slow2, respectively. When the sensors were expressed in HEK293T cells, lower decay t 1/2 -s of 6.9, 115, and 234 ms were measured for iGlu3Fast, iGlu3Slow1 and iGlu3Slow2, respectively, in accordance with reduced K d -s of ∼200 μM, 6.4 and 8.4 μM, respectively. Both iGlu3Fast and iGlu3Slow2 reported spontaneous activity with high sensitivity in organotypic hippocampal slices by 2-photon imaging. Strikingly, different kinds of glutamate transient were detected by the fast- and slow-decay sensors indicating that the rapid kinetics and increased fluorescence dynamic range make iGlu3Fast an excellent candidate for imaging high-frequency glutamate release at synapses while iGlu3Slow2 has the potential for mesoscale imaging of brain activity to record global events with high sensitivity. Acknowledgments: BBSRC BB/S003894 to K.T. and T.C.
Tran et al. (Sun,) studied this question.