Table 1.

Nonadditivity of the perturbations at E395 and T469 evaluated using FMC

Mutant	V1/2 (±SEM)	z (±SEM)	n	ΔGapp (±SEM)
	mV			kcal
WT	−21.8 (±1.4)	2.5 (±0.07)	4	−1.3 (±0.09)
E395A	−59.3 (±0.6)	7.7 (±0.17)	4	−10.5 (±0.25)
T469A	−36.5 (±1.3)	3.7 (±0.32)	4	−3.1 (±0.29)
E395A-T469A	−50.8 (±1.0)	4.1 (±0.28)	3	−4.8 (±0.34)

The V_1/2 values of the G-V curves of the inactivation removed Shaker K_V (inactivation removed) channel, and the mutants are similar to those reported previously (Yifrach and MacKinnon, 2002). However, our fitted Boltzmann slopes (z) observed in this study are consistently lower than those reported previously. This can be attributed to the differences in the solutions used for functional measurements of the tail currents (110 mM NMG-MES/10 mM K-MES vs. 58 mM NaCl/40 mM RbCl). As a result of this difference in slopes, the ΔΔG_FMC calculated in this study (+7.5 ± 0.52 kcal/mol) is lower than that reported previously.