Fig. 1.
Characterization of the YECL motif in GABAAR γ2-subunits that binds AP2. (A) YECL-pep, but not AECL-pep, interacts directly with [35S]-labeled μ2–AP2 (residues 158–435 containing the Yxxφ motif C-terminal-binding domain). A [35S]-labeled truncated construct lacking the Yxxφ motif-binding pocket (residues 158–407) no longer binds YECL-pep. (B) YECL, but not AECL, beads associate with purified bacterially expressed His-μ2 (residues 156–435). YECL beads show reduced association with an identical His-μ2 fusion protein containing W421 mutated to A. (C and D) SPR analysis of the binding of YECL-pep to purified His-μ2 reveals a Kd of 42.2 nM. (C) Sensograms of binding His-μ2 to YECL-pep performed on a BIACORE 2000. His-μ2 was injected at concentrations from 62 nM to 2 μM (lower to upper curves) over immobilized YECL-pep. The change in SPR signal during association and dissociation is shown in colored curves. Black bars are report points set on the sensograms in the steady-state region of the curve. (D) Plot of steady-state binding levels (Req) against concentrations of μ2 and fit to steady-state affinity model. (E–J) Functional consequences of blocking γ2-subunit interaction with AP2 on inhibitory synaptic responses. (E) Plot of normalized mIPSC amplitude as a function of time in cells dialyzed with YECL-pep and control AECL-pep. YECL-pep increases mIPSC amplitude. (F and G) Representative traces (F) and cumulative plots (G) from the 3rd and 57th minutes in cells dialysed with YECL-pep. (H and I) Representative traces (H) and cumulative plots (I) from the 3rd and 57th minutes in cells dialysed with control AECL-pep. (J) Bar plot summary showing the differential effects of YECL-pep and control AECL-pep on mIPSC amplitude and frequency.