title: Direct binding of GABAA receptor β2 and β3 subunits to gephyrin.
abstract: GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABA(A) R) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)-associated protein gephyrin has been shown to be essential for the clustering of a subset of GABA(A) R. Despite recent progress in the field of gephyrin-dependent mechanisms of postsynaptic GABA(A) R stabilisation, the role of gephyrin in synaptic GABA(A) R localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABA(A) R α1, α2, β2 and β3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20-fold lower affinity of the β2 loop to gephyrin as compared to the GlyR β loop-gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well-established use of chimaeras of GlyR α1 that harbour respective gephyrin-binding motifs derived from the different GABA(A) R subunits. These studies identify a novel gephyrin-binding motif in GABA(A) R β2 and β3 large cytoplasmic loops.
authors.affiliation: Department of Chemistry, Institute of Biochemistry, University of Cologne, Cologne, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kowalczyk
authors.forename: Aline
authors.initials: A
authors.lastname: Winkelmann
authors.forename: Birthe
authors.initials: B
authors.lastname: Smolinsky
authors.forename: Benjamin
authors.initials: B
authors.lastname: Förstera
authors.forename: Ines
authors.initials: I
authors.lastname: Neundorf
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
authors.forename: Jochen C
authors.initials: JC
authors.lastname: Meier
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011963:Receptors, GABA-A; C073441:gephyrin