Rational design of optical sensors for probing neurotransmitter dynamics

Abstract

Synaptic plasticity – the ability of the synapse to strengthen or weaken in response to external stimuli is one of the most fundamental aspects of neurological function. Improving our understanding of the neurotransmitter dynamics that govern synaptic plasticity will also improve our appreciation of the wider neural system, giving us the tools we need to recognize and treat neurological disorders. Multiple molecules play a role in synaptic plasticity. The amino acid neurotransmitters L-arginine, glycine and Dserine are of particular interest. L-arginine functions as the sole precursor to nitric oxide, an integral secondary messenger, influencing the release and reuptake of the neurotransmitter L-glutamate from neurons. No dynamic behaviour for L-arginine is explicitly documented; however, there are strong indications that it plays such a role in synaptic plasticity. D-serine and glycine are known transmitters that bind to NMDA receptors as co-agonists alongside L-glutamate. Their contribution to synaptic plasticity is appreciated, but the spatio-temporal dynamics of their roles are not clearly defined. The study of neurotransmitter dynamics is often impaired by the absence of a suitable sensory device or method. Optical sensors present a tool with which to study dynamic processes in vivo and in real time, providing high spatio-temporal resolution information about a specific neurotransmitter in an experimental setup that can be used in conjunction with existing electrophysiological methods. An optical sensor capable of detecting these transmitters in a biological context would provide the means to study the regulation of synaptic plasticity relating to nitric oxide and furthermore, to understand the release, uptake and localization of glycine and D-serine in the brain, in relation to the excitatory process of long term potentiation. Understanding these elements would advance our understanding of synaptic plasticity and how it mediates learning and memory processes. The development of any sensor requires the presence of a suitable recognition element specific for its target ligand, such as a solute binding protein. Although nature has evolved binding proteins for an array of molecules and environments, these do not always meet experimental requirements. Current L-arginine sensors based on extant binding proteins suffer from low thermostability, making long term experiments in physiological conditions difficult and precluding the acquisition of useful data. Currently, no optical D-serine or glycine sensors exist. In this work we describe the engineering and application of multiple genetically encodable optical sensors for amino acid neurotransmitters. Firstly for L-arginine, cpFLIPR (circularly permuted Fluorescent Indicator Protein for arginine), based on an ancestral solute binding protein scaffold, secondly for glycine, based on a GABA/glycine binding protein and finally a D-serine specific optical sensor, SERIOS (SERIne Optical Sensor) engineered from a D-alanine binding protein. Finally, a predictive model – Rangefinder – was developed for the construction of semi-synthetic sensors. The application of Rangefinder was illustrated by the creation of novel sensors for maltose, sialic acid and L-arginine. The methods and designs used during these studies build on previous work to create optical sensors and provide new insights and methodologies for future development of optical probes for new ligands of interest.