Figure 2.

Residues E321C/E324C at the entrance to the inner cavity are accessible to the intracellular solution. (A–C) Representative records of outward currents through single BK channels held at +180 mV before and after applications of thiol reagents to wt and mutated channels. The dashed lines show the open and closed current levels of wt channels. Current levels after intracellular application of MPA⁻, MBB⁻, or MTSET⁺ to wt channels followed by thorough washing (A) were unchanged, whereas application and wash to E321C (B) or E324C (C) channels changed single-channel current levels after treatment with MPA⁻ or MTSET⁺. (D and E) Plots of i_out versus voltage for the indicated treatments for E321C (D) and E324C (E) channels. Treatment and wash of the thiol agents to wt channels had no effect on i_out and are not plotted. In this and subsequent plots, the dashed lines are cubic spline fits, and the absence of visible error bars indicates that the SEM is less than the symbol size. Data are from three to five different oocytes for each plotted symbol. Positions E321C and E324C are both accessible to the ion conduction pathway from the intracellular side with equivalent effects of thiol reagents at these positions on i_out. 150 mM K⁺_i.