Figure 3. Labeling GABA_A receptors with ortho-propofol diazirine.

With both α₁β₃ heteromers and β₃ homomers only a single amino acid, H267, was labeled. (a) MS2 data for the peptide 260-TMTTINTHL-268 labeled with the protiated ortho-propofol diazirine (black) and the deuterated ortho-propofol diazirine (red) superimposed. (b) Fragmentation diagram showing that the MS2 data are consistent with the ortho-propofol diazirine labeling the histidine side chain which subsequently rearranges as internal ions H* and TH* following fragmentation. (c) A view of our proposed propofol binding site seen from the center of the pore. Two neighboring subunits are shown in yellow and blue and a single ortho-propofol diazirine molecule is shown associated with the yellow subunit. (d) A view from the extracellular side of the receptor, but with the extracellular domain removed for clarity. The β₃ homopentamer is shown with five equivalent ortho-propofol diazirine molecules bound. (e) A surface representation of our proposed propofol binding pocket with an ortho-propofol diazirine molecule sitting in the pocket close to H267 which is the amino acid that is photolabeled. (f) Electrophysiological data from α₁β₃ heteromers showing the effects of the mutations β₃-H267A and β₃-F221W on the GABA apparent affinity (n=21), (g) the extent to which 3 μM ortho-propofol diazirine potentiates a GABA-evoked chloride current (at the GABA EC₆₀) (n=4–10) and (h) the extent to which 10 μM ortho-propofol diazirine directly activates the receptor (n=4). All error bars are standard errors of the mean.