Gephyrin selective intrabodies as a new strategy for studying inhibitory receptor clustering.
The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.
Zacchi
Paola
P
Neuroscience Programme, International School for Advanced Studies, Via Beirut 2-4, 34014 Trieste, Italy. zacchi@sissa.it
Dreosti
Elena
E
Visintin
Michela
M
Moretto-Zita
Matteo
M
Marchionni
Ivan
I
Cannistraci
Isabella
I
Kasap
Zeynep
Z
Betz
Heinrich
H
Cattaneo
Antonino
A
Cherubini
Enrico
E
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D018009:Receptors, Glycine / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D002352:Carrier Proteins; D018690:Excitatory Amino Acid Agonists; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D018009:Receptors, Glycine; D011993:Recombinant Fusion Proteins; C073441:gephyrin; D049452:Green Fluorescent Proteins; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D005998:Glycine