Zachariassen, L. G. | bioRxiv Science

bioRxiv2025

FLIM-FRET Imaging of AMPA Receptors: New Principle for Subtype-Specific Elucidation

-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory neurotransmission, and their subunit composition (GluA1-4) critically shapes synaptic strength and plasticity. Discriminating AMPAR subtypes at the molecular level remains challenging with conventional techniques. Here, we applied Forster resonance energy transfer (FRET) combined with fluorescence-lifetime imaging microscopy (FLIM) to resolve subtype-specific AMPAR assemblies. Cyan fluorescent protein (CFP) and HALO domain were genetically introduced into GluA1-3 subunits to make intrareceptor FRET pairs. Self-labeling protein (SLP) domains such as SNAP and HALO were used with cell-impermeable substrates to selectively label surface-expressed receptors. This approach identified di-heterotetrameric GluA1/2 and GluA2/3 assemblies at the HEK293T cell membrane, whereas GluA1/3 failed to form di-heteromers. These findings demonstrate the FLIM-FRET method as a tool for differentiating AMPAR subtypes in living cells, providing a foundation for studying subtype-specific receptor organization in excitatory synapses.

neuroscience↗

bioRxiv2022

Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and two additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.

biochemistry↗