Figure 6.

Pore exposure and residue proximity inferred by site effects on single-channel current or disulfide bond formation. (A) Single WT ΔK_2PØ channels or EE142,149QQ ΔK_2PØ channels with no other change or the indicated third mutation studied at 60 and −100 mV in on-cell patches with 140-mM KCl solution in the bath and pipette (see Materials and methods). Unitary currents (i) were determined from 2 to 8.5 min of recording from two to six patches sampled at 20 kHz, filtered at 5 kHz with one to nine channels per patch (median = 2), and noted ±SEM. Exemplar currents were filtered at 500 Hz. Open (O) and closed (C) levels are indicated. (B, left) Predicted topology of ΔK_2PØ indicating the location of F199 and F239. (middle) Whole cell currents studied at 30 mV. The channels studied had the native external cysteine in the first external loop altered to serine (C73S). Application of 0.5 mM DTNB reduces C73S F199C F239C ΔK_2PØ current (black circles), and 5 mM DTT reverses the effect. Current of ΔK_2PØ C73S F199C (open diamonds) or C73S F239C ΔK_2PØ (open triangles) is not affected by exposure to DTT or DTNB. Plot is normalized current for single cells; every 20th data point. (right) Current ± SEM at 30 mV after application of 5 mM DTT (open bars) or 0.5 mM DTNB (closed bars) normalized to the amplitude of the same cell without treatment for ΔK_2PØ channels with the following changes: C73S and F199C; C73S and F239C; C73S and F199C and F239C (n = 2–4 cells).