Tracking cell surface mobility of GPCRs using α-bungarotoxin-linked fluorophores.
GABA(B) receptors are G-protein-coupled receptors (GPCRs) that are activated by GABA, the principal inhibitory neurotransmitter in the central nervous system. Cell surface mobility of GABA(B) receptors is a key determinant of the efficacy of slow and prolonged synaptic inhibition initiated by GABA. Therefore, experimentally monitoring receptor mobility and how this can be regulated is of primary importance for understanding the roles of GABA(B) receptors in the brain, and how they may be therapeutically exploited. Unusually for a GPCR, heterodimerization between the R1 and R2 subunits is required for the cell surface expression and signaling by prototypical GABA(B) receptors. Here, we describe a minimal epitope-tagging method, based on the incorporation of an α-bungarotoxin binding site (BBS) into the GABA(B) receptor, to study receptor internalization in live cells using a range of imaging approaches. We demonstrate how this technique can be adapted by modifying the BBS to monitor the simultaneous movement of both R1 and R2 subunits, revealing that GABA(B) receptors are internalized as heteromers.
Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom.
Saad
S
Hannan
Megan E
ME
Wilkins
Philip
P
Thomas
Trevor G
TG
Smart
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D058948:Cell Tracking / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods; D018080:Receptors, GABA-B / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D002038:Bungarotoxins; D005456:Fluorescent Dyes; D018080:Receptors, GABA-B