Figure 6. Pharmacological specificity of [3H]pTFD-di-iPr-BnOH photolabeling of α1β3γ2 GABAARs. A & B.
Aliquots of GABAARs were photolabeled with 1.5 μM [3H]pTFD-di-iPr-BnOH in the presence of 100 μM bicuculline (Bic; resting state receptors) or 300 μM GABA (desensitized receptors) in the absence of other drugs and in the presence of 100 μM propofol, 60 μM R–mTFD-MPAB (MPAB), 100 μM pTFD-di-iPr-BnOH, 300 μM etomidate (Etom), or 30 μM allopregnanolone (3,5-P). After photolabeling, receptor subunits were resolved by SDS-PAGE and 3H incorporation was determined by fluorography (A, except that lane 1 is a representative Coomassie Blue stained gel lane) or by liquid scintillation counting of excised subunits (B). Indicated on the left are the mobilities of the molecular mass markers and the calculated mobilities of the GABAAR subunit bands (α1, 56 kDa; β3, 59/61 kDa; with the γ2 subunit distributed diffusely in the three bands). B. In parallel with the fluorogram, 3H incorporation in the excised α (56 kDa) and β (59/61 kDa) subunit gel bands was determined by liquid scintillation counting. The means ± 1/2 range are plotted from 2 gels. C. The concentration dependence of inhibition of β subunit photolabeling by pTFD-di-iPr-BnOH (closed circles), propofol (open circles) or R–mTFD-MPAB (open squares). Non-specific photolabeling (Bns) was determined in the presence of 300 μM etomidate and 60 μM R-mTFD-MPAB. For inhibition by pTFD-di-iPr-BnOH and propofol, which was determined for [3H]pTFD-di-iPr-BnOH at 2 μM, Bns was 25 ± 3%, (N = 4, dashed line). For R–mTFD-MPAB inhibition, determined at 3 μM [3H]pTFD-di-iPr-BnOH, Bns was 42 ± 8% (N = 5). For each independent experiment, data were normalized to the control condition, and the plotted data are the means (± SD) from the pooled independent experiments. The pooled data from the independent experiments were fit to Eq. 3 (see Materials and Methods) with IC50 as a variable parameter. Parameters for the fits and the number of independent experiments (N) are tabulated in Table 1.