Figure 4.
Intracellular loading can result in potentiation. A, Resurgent steroid effects after cyclodextrin wash. A1, Slow direct gating of GABAA receptors in response to 1 μm ACN in a transfected HEK cell. The cell was rapidly perfused with drug-free saline after ACN removal but nevertheless had a slowly decaying off response, similar to that previously described for neurons and the natural neurosteroid 3α5αP (Shu et al., 2004). A2, In the same cell on another application of ACN, the offset current was reversibly blocked by bicuculline (bic), which is a noncompetitive antagonist with respect to steroid (Ueno et al., 1997). Therefore, in the case of bicuculline, a large part of the resurgence results from bicuculline unbinding before steroid departure. A2, A3, The offset current was partly inhibited by a brief wash with 500 μm γ-cyclodextrin (CDX), which facilitates the removal of steroid (Shu et al., 2004). The resurgent current (arrows) indicates that the replenishment of receptor-accessible steroid, possibly from the intracellular pools, which are inaccessible to cyclodextrin. To facilitate comparison of offset, the dotted trace represents a replot of the control trace in A1, scaled to account for slight rundown between sweeps. Note that the resurgence is less complete than for bicuculline and less than expected from baseline decay (dotted traces). B, Effects of intracellular steroid loading. The currents presented show representative data from a total of four cells under control conditions and five cells loaded with steroid. B1, Response to 1μm GABA in a control transfected HEK cell. Note that the GABA response is insensitive to 500 μm cyclodextrin. B2, In a cell loaded with 50 μm ACN through the whole-cell pipette, 1μm GABA gated a larger response, which was partly sensitive to 500μm γ-cyclodextrin. C1, Channel activity in an inside-out membrane patch (50μm GABA in the pipette), to which 1 μm ACN was applied by bath exchange to the intracellular face. C2, Summary of channel effects. Channel openings are shown downward. The open times were 0.42 ms (48%), 4.2 ms (15%), and 23.3 ms (37%). The closed times were 0.28 ms (48%), 1.4 ms (42%), and 14.5 ms (10%).