Figure 4. Br-bTBA and bbTBA block closed MthK more slowly than TPeA.

(a) Relative Tl⁺ flux rates as a function of Br-bTBA and bbTBA (structures inset) incubation with closed MthK channels. Blocker equilibrations were fit to exponential functions, τ_Br-bTBA = 9 ± 3 seconds, τ_bbTBA = 32 ± 7 seconds (Eq. 10, black and red solid lines, respectively). The fitted blocker time course for TPeA (from Fig. 3b) is shown for comparison (black dashed line). (b) Relative Tl⁺ flux rates versus incubation time with open MthK for 1 (black squares) and 3 μM (red circles) bbTBA. The results were simultaneously fit to Eq. 11 (lines; K_D^open = 2.1 ± 0.2 μM, k_on^open = 17 ± 2 μM⁻¹ s⁻¹). (c) Closed state Br-bTBA dose response curve for 45 s blocker incubations prior to activation of MthK by 17.2 mM Ca²⁺ (black squares). The results were fit to the Hill equation (red line; K_D^closed = 2.5 ± 0.2, n_H = 1.16 ± 0.09). Dose response for open state Br-bTBA block measured during the instrument dead time (grey circles). Grey line has no theoretical meaning. Symbols are the mean ± s.d. from at least three (only two for bbTBA data in b) independent measurements.