Fig. 2.

LRET measurements of LBT-tagged BK constructs. (A) Representative DO emission recording from an oocyte expressing the S0 construct. The red solid line represents a three-exponential fit. The slowest component with a time constant of ≈2.5 ms (red dotted line) corresponds to the decay from Tb³⁺ bound to LBT (Inset). (B) SE of the acceptor recorded after addition of IbTX-bodipy from the same oocyte of A. (Inset) Attached fluorophore acts as an LRET acceptor. Gray traces in A and B correspond to DO and SE control experiments, respectively, using WT BK α-subunit (α-BK). (C) Representative SE recorded from NT, S0, S1, and S2 constructs exhibited characteristic decay kinetics. Because there are four donors and one acceptor per channel, SE decays are composed of four energy transfer-based exponential components. Faster SE kinetics indicate that the four donors are positioned in closer proximity to the acceptor. a.u., arbitrary units.