Figure 3.

Ba²⁺ block of WT rSK2, open state. (A) An inside-out membrane patch containing WT rSK2 channels was voltage clamped and the voltage ramped from –80 to 40 mV (80 mV/s) in asymmetrical K⁺ (2 K_out, 150 K_in) and 2 μM Ca²⁺ (fully open) either without (Cont) or with 1 μM Ba²⁺ (1 μM Ba) to illustrate that block is strongly voltage dependent. (B) Consecutive 10-s voltage steps from −80 to 0 mV in the presence of increasing concentrations of Ba²⁺ (in μM: 0, 0.01, 0.1, 0.3, 1, 3, 1,000) are overlaid to demonstrate the WT SK2 channel response to Ba²⁺ applied to the intracellular side of the membrane. (C) The average current amplitude over the final 1 s of each voltage step in B was normalized, plotted against Ba²⁺ concentration, and the points fitted with a single binding isotherm (see Materials and methods), yielding an apparent dissociation constant (IC₅₀) of 997 nM. Inset, plot of inverse time constant determined from fits of a single exponential to traces in B versus Ba²⁺ concentration. Line represents a linear fit to data yielding a slope 1.4 × 10⁶ M⁻¹s⁻¹ and an intercept of 1.1 s⁻¹.