Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes

Abstract

We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGlu h , iGlu m , and iGlu l cover a broad range of K d -s (5.8 mM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a K d of 9.7 mM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGlu u and iGlu f had monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlu m and iGlu l , the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGlu h and Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusionlimited rate constant to iGlu h and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding.

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

“…To better understand how kinetic properties of the indicator impact these signals, here we consider a simple reaction-diffusion system in which glutamate transients are released from point sources, and glutamate subsequently interacts with the expressed indicator. Consistent with our data and previous work (e.g., (50)), we model the indicator as having three states, described below. We model the extracellular interface as a 2D surface.…”

“…The above simulations suggest that the spatial specificity of existing iGluSnFR variants is limited by ON rate saturation, which is determined by kinetic parameters that vary substantially among existing variants (22,50). These kinetic parameters are easily measured, and can therefore be engineered.…”

“…iGluSnFR and its variants exhibit saturating ON rates, in which the formation of fluorescent species cannot exceed a maximum rate v max , presumably due to a rate-limiting conformational change (22,50). ON rate saturation is characteristic of a kinetic model with at least 3 states, in which ligand binding (k 0-1 ) rapidly forms a bound but dim species (S1), followed by a slower transition ( The effects of K M and v max on spatial specificity are distinct (panel F).…”

“…Since the ligand concentration close to the release site is always larger than that further away, the ratio of the indicator signal near the release site to that farther away is at most equal to the ratio of the concentrations. iGluSnFR and its variants exhibit saturating ON rates, in which the formation of fluorescent species cannot exceed a maximum rate v max , presumably due to a rate-limiting conformational change (22,50). ON rate saturation is characteristic of a kinetic model with at least 3 states, in which ligand binding (k 0-1 ) rapidly forms a bound but dim species (S1), followed by a slower transition ( The effects of K M and v max on spatial specificity are distinct (panel F).…”

Entering a new era of quantifying glutamate clearance in health and disease

“…These SF-iGluSnFR variants have higher signal-to-noise ratios, allowing for improved transient detection in single spines compared to original iGluSnFR. In addition, variants iGlu h , iGlu m and iGlu l were recently designed to cover a wide affinity range, with Kd values ranging from low micromolar to tens of millimolar (Coates et al, 2020). Additional versions have been designed with a yellow-light excitable red fluorescent protein, termed R-iGluSnFR and R ncp -iGluSnFR (Wu et al, 2018).…”