Comparing the effect of S367 and M368 mutations on activation and deactivation times. Bar graphs describing the effect of varying the solvation energy of the residue at positions 367 and 368 on the channel activation (t_on) and deactivation times (t_off). (A) Results demonstrating that mutations at S367 that caused an increase in Pomax and t_off (B) tend to accelerate channel activation with smaller t_on values relative to WT. The mutation S367E led to a faster t_on, despite no significant effect on Pomax or t_off compared with WT. (B) Effect on the channel deactivation time t_off of mutating S367 by residues of equivalent or higher transfer energy to water. Although the S367A and S367E mutations had no impact on t_off, significant increases were seen with the S367T (t_off = 176 ± 7 ms; n = 4), S367C (t_off = 234 ± 25 ms; n = 7), S367L (t_off = 289 ± 21 ms; n = 3), and S367W (t_off = 799 ± 22 ms; n = 5) mutants. The inset illustrates the correlation between the relative change in t_off computed as ln(t_{off_mutant}/t_{off_WT}) and the solvation energy for water to n-octanol transfer (Wimley et al., 1996). With a Pearson correlation coefficient of 0.90, for nonpolar residues, this analysis confirms that hydrophobic interactions at 367 of KCa3.1 play a critical role in setting Pomax. Substituting to a charged residue (S367E) yielded faster t_off values compared with t_off measured for nonpolar residues of equivalent surface area (Leu). (C) Bar graph summarizing the effect of mutating the CaM-anchoring residue M368 on the channel activation time t_on. With the exception of the M368W mutant, higher t_on values correlate with an increase in free energy of transfer to water. (D) Bar graph demonstrating that in contrast to the activation time t_on, mutating M368 had no impact on the deactivation time t_off and thus on the stability of the channel open configuration.