Illuminating Ionotropic Glutamate Receptors: Characterization of Fluorescent Antagonists Based on Polyamine Toxins

Ionotropic glutamate (iGlu) receptors, including the NMDA and AMPA subfamilies, mediate fast excitatory synaptic transmission in the mammalian central nervous system. NMDA and AMPA receptors play critical roles in learning and memory and are implicated in multiple neurological and psychiatric diseases. Argiotoxins (exemplified by the prototypical ArgTX-636) are so-called polyamine toxins isolated from the venom of the orb weaver spider Argiope lobata. Argiotoxins consists of an aromatic head group coupled via an amino acid linker to a polyamine moiety and act as open-channel blockers of the iGlu receptor ion channel with nanomolar affinity. Structure-activity studies of synthetic argiotoxin analogs have shown that modification of the polyamine tail can tweak NMDA versus AMPA selectivity (REF NELSON ET AL). Furthermore, the aromatic head group can tolerate substantial modification without loss of affinity.The aim of the present study is to develop NMDA- and AMPA selective argiotoxin analogs containing fluorescent moieties to enable imaging studies of native iGlu receptors and to serve as potential FRET donor or acceptor ligands. Specifically, a series ArgTX-636 analogs with fluorescein, coumarin and boron-dipyrromethene (BODIPY) derived fluorophores incoporated at or replacing the headgroup moiety were synthesized and characterized at recombinant AMPA and NMDA receptors expressed in Xenopus Laevis oocytes (GluA1 and GluN1/2A, respectively). Several analogs were found to posses high-affinity NMDA and/or AMPA receptor binding while maintaining desirable fluorescent properties.