Fig. 1.

Analyzing the interaction of GABA_AR α subunits with collybistin. a Cartoon showing the purified proteins used for biochemical studies of α-subunit interaction with gephyrin and CB. b Receptor subunits were subject to NAGE (pockets mark sites of protein application) followed by coomassie staining, revealing that α-subunit loops slowly migrate to the cathode. CB-SH3 alone or mixed with a threefold excess of GABA_AR intracellular domains reveals that α2L markedly alters the migration of SH3 (red arrow). c SEC analysis was used to explore the behavior of CB-SH3 alone or when mixed with equimolar amounts of α1L, α2L, or α3L. Fusion proteins were detected in eluates using absorbance at 280 nM and coomassie staining after SDS-PAGE (insets), verifying the presence of the SH3 domain at lower elution volumes in the fraction containing α2L. d CB-SH3 was titrated against α1L and α2L. The measured binding enthalpies are plotted as a function of the molar ratio of the SH3 domain to the GABA_AR α1 and α2 loops. e GephE and GephE + CB-SH3 were subject to NAGE. The migration of the respective proteins is indicated. In presence of the SH3 domain the migration of gephyrin is not altered indicating that the proteins do not interact on NAGE (left panel). GephE in the presence of a fivefold molar excess of α2L is partly retained in the pocket and partly migrates towards the anode (lane 1 right panel). Addition of increasing amounts of the SH3 domain to the GephE-α2L complex allows more gephyrin to enter the gel and migrate towards the anode. At the same time SH3 binding to α2L retains SH3 in the pocket. This indicates that SH3 and gephyrin compete for α2 binding. f CB-SH3 was titrated against α2L alone or in combination with GephE, and the measured binding enthalpies are plotted