Figure 5.
α1(K104C)β2γ2(D75C) receptors spontaneously cross-link and reform this cross-link after DTT application. A, illustration depicting the disulfide bond that spontaneously cross-links the α1 and γ2 subunits of the α1(K104C)β2γ2(D75C) GABAAR, is broken after DTT application, and slowly reforms over time. B, effect of PMTS application on currents elicited by the GABA EC5–10 of WT, α1(K104C)β2γ2, α1β2γ2(D75C), and α1(K104C)β2γ2(D75C) GABAA receptors. The change in GABA EC5–10 currents by PMTS was decreased in single mutant receptors both before (F(3,18) = 14.56, p < 0.05) and after (F(3,18) = 45.45, p < 0.001) DTT application compared with those seen in WT receptors. The change in EC5–10 currents produced by PMTS was not significantly altered after DTT application to WT or single mutant receptors but did significantly change in double mutant receptors (F(3,37) = 41.698, p < 0.001)). A one-way ANOVA showed a significant effect of time after DTT treatment (pre-DTT treatment, 5 min after, and 60 min after) on α1(K104C)β2γ2(D75C) GABAAR (F(2,13) = 108.363, p < 0.001), and a Tukey's post hoc test showed a significant difference between pre-DTT and 5 min after DTT application and a significant difference between 5 min after DTT and 60 min after DTT (***, p < 0.001; each symbol represents an oocyte, and each bar represents the mean response. Error bars represent the S.E.). C, sample tracing showing spontaneous reformation of the α1–γ2 intersubunit disulfide bond in the α1(K104C)β2γ2(D75C) GABAAR. The GABA EC5 measured in the oocyte before DTT application was 3 μm GABA, but after DTT application 3 μm GABA elicited a much smaller response. After ∼60 min, the response to 3 μm GABA had returned to pre-DTT levels. D, time courses of EC5 values plotted for five oocytes expressing α1(K104C)β2γ2(D75C) receptors returning to their pre-DTT values. This can also be interpreted as the time required to reform the disulfide bond after DTT application. The average time to return to half of the pre-DTT EC5 was 26.9 ± 2.5 min.