TABLE I.

Parameters of bbTBA Blockade

	Macroscopic		Microscopic
	Kd vs. V	Frac. block	Kinetics	Steady state	FINAL MODEL
	(Fig. 1 C)	(Fig. 8 C)	(Fig. 5)	(Fig. 5 C)
Kd(0 mV) μM	9.5	8.3, 8.1, 10.1a	5.5	4.8	8–10
zδ total	0.15	0.13, 0.16, 0.17a	0.11	0.14	0.15
kON(0 mV)     M−1 s−1			1 × 108		0.625 × 108
zδON			0.05		0.05
kOFF(0 mV)     s−1			562		562
zδOFF			0.07		0.1

The dissociation constant (K_d) reflects the concentration of bbTBA required for half-maximal blockade. The charge carried by bbTBA (z = +1) multiplied by the total electrical distance δ together give zδ_total, an estimate of the total voltage dependence of block. The second order association rate for block at 0 mV (k_ON (0 mV)) and the charge associated with blocker binding zδ_ON, as well as the rate and voltage dependence of blocker dissociation (k_OFF (0 mV) and zδ_OFF), were calculated from microscopic kinetic measurements as described in Figs. 4 and 5. Macroscopic K_d and zδ were obtained from fitting K_d values (obtained from Hill fits) plotted as a function of voltage (Fig. 1 C) with Eq. 2, or fitting fractional unblock plotted as a function of voltage (Fig. 8 C) to Eq. 3. Microscopic K_d and zδ were measured from fits to Eq. 2 of K_d vs. voltage relationships (Fig. 5 C), where K_d was obtained from either kinetic measurements (Fig. 4 C) or steady state P_o (Fig. 4 D). Blocker rates and associated zδ values were obtained from fitting plots of either the on or off rates from dwell time histograms vs. voltage (Fig. 5, A and B) with Eq. 2. A final model was constructed that describes all measurements in this study; this model was used in modeling block (Figs. 1, 3, 5, 8, 10, and 11; see Materials and methods).

^aValues given are for low, medium, and high P_o data sets, respectively.