Genetically encoded intrabody probes for labeling and manipulating AMPA-type glutamate receptors

Dean J. Kareemo, Christina S. Winborn, Samantha S. Olah, Carley N. Miller, JungMin Kim, Chelsie A. Kadgien, Hannah S. Actor-Engel, Harrison J. Ramsay, Austin M. Ramsey, Jason Aoto and Matthew J. Kennedy ()
Additional contact information
Dean J. Kareemo: Anschutz Medical Campus
Christina S. Winborn: Anschutz Medical Campus
Samantha S. Olah: Anschutz Medical Campus
Carley N. Miller: Anschutz Medical Campus
JungMin Kim: Anschutz Medical Campus
Chelsie A. Kadgien: Anschutz Medical Campus
Hannah S. Actor-Engel: Anschutz Medical Campus
Harrison J. Ramsay: Anschutz Medical Campus
Austin M. Ramsey: Anschutz Medical Campus
Jason Aoto: Anschutz Medical Campus
Matthew J. Kennedy: Anschutz Medical Campus

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: Abstract Tools for visualizing and manipulating protein dynamics in living cells are critical for understanding cellular function. Here we leverage recently available monoclonal antibody sequences to generate a set of affinity tags for labeling and manipulating AMPA-type glutamate receptors (AMPARs), which mediate nearly all excitatory neurotransmission in the central nervous system. These antibodies can be produced from heterologous cells for exogenous labeling applications or directly expressed in living neurons as intrabodies, where they bind their epitopes in the endoplasmic reticulum and co-traffic to the cell surface for visualization with cell impermeant fluorescent dyes. We show these reagents do not perturb AMPAR trafficking, function, mobility, or synaptic recruitment during plasticity and therefore can be used as probes for monitoring endogenous receptors in living neurons. We also adapt these reagents to deplete AMPARs from the cell surface by trapping them in the endoplasmic reticulum, providing a simple approach for loss of excitatory neurotransmission. The strategies outlined here serve as a template for generating similar reagents targeting diverse proteins as more antibody sequences become available.

Date: 2024
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DOI: 10.1038/s41467-024-54530-5

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