Coupling energies determined by double mutant cycle analysis for the channel t_on and t_off. ΔG was calculated as KT ln(t_{off_mutant}/t_{off_wt}) or KT ln(t_{on_double_mutant}/t_{on_mutant}), where K is the Boltzmann constant and T is the absolute temperature. The coupling energy ΔΔG was computed as (ΔG_{mutation1–mutation2} + ΔG_WT–WT) − (ΔG_{mutation1–WT} + ΔG_{mutation2–WT}). Differences in free energies are given in kcal/mol. (A) The mutation Q41A on CaM strongly affected the potency of the S367W mutation to increase t_off. (B) Absence of coupling energy for channel activation between E363 and R352 despite the prediction of strong electrostatic interactions as a result of E363 facing R352 on the adjacent subunit. (C) Importance of the Lys residue at position 75 on CaM to the decrease in activation time caused by the R362E mutation. The charge neutralization K75A resulted in the charge reversal R362E being ineffective in modulating the channel activation process. These results also indicate that the R362 (KCa3.1)–K75 (CaM) interaction contributes to the energy barrier accounting for channel activation, while supporting the structural model presented in Fig. 1. (D) Importance of the Glu residue at position 47 on CaM to the charge neutralization effect on t_on seen with the K360C mutation. The mutation E47A on CaM compromised the potency of the mutation K360C to increase t_on, suggesting a contribution coming from electrostatic interactions. (E and F) Functional coupling between E363 and S367 in regulating the channel activation (E) and deactivation (F) times. In E, the drastic increase in activation time caused by the charge reversal E363R mutant was significantly reduced when coupled to the mutation S367C, an effect attributable to the substitution S367C stabilizing the channel open configuration thus facilitating channel activation. In F, the mutation S367C can no longer be translated into an increased deactivation time when coupled to the E363R mutation, an effect likely attributable to the mutation E363R increasing the energy barrier to form a functional channel, and thus facilitating the channel exit from a conducting mode (see Eq. 1).