Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes

Abstract

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. 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: affinity variants of iGluSnFR termed iGluh, iGlum and iGlul covering a range of Kd-s (5.8 M, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with a Kd of 9.7 M are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluu and iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluh and Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.\n\nSTATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.