Development of an Activity Dependent, Photochemically-Released Tool for the Study of Calcium Permeable AMPA Receptors

Abstract

The ability to observe the movement of neurotransmitter receptors in and around a synapse could provide crucial new information to our understanding of synaptic plasticity, the process that likely underlies memory formation. We have developed a new class of fluorescent probes designed to target fluorophores to natively-expressed neuronal receptors. This strategy allows for receptors to be covalently-tagged and tracked in a non-perturbed state; thus allowing for visualization of complex neuronal processes. Specifically, calcium-permeable, non-NMDA glutamate receptors (CP-AMPARs) expressed in hippocampal neurons can be targeted with this novel tri-functional molecule. CP-AMPARs have recently been shown to play a role in some forms of synaptic plasticity, aiding such processes as long-term potentiation and depression, but their basal location around the synapse remains unknown. In brief, our molecular design employs a use-dependent polyamine ligand which targets only the receptors receiving glutamatergic input at the time of labeling, a promiscuous electrophile for covalent bond formation with a nucleophillc sidechain amino acid on the channel, and a fluorophore for visualization. Bioconjugation of this molecule results in stable covalent bond formation between the probe and the target receptor. An additional aspect of our first generation probes is that the ligand is connected to the remainder of the probe with a photolabile linker, thus allowing the receptor to re-enter the non-liganded and native state.