Relative difference in the peak value of the free energy barrier during R4 transition for all hydrophobic core residues mutated to alanine, compared to the value for WT.

The standard error for the WT is the absolute standard error. A decrease or increase is defined by the cutoff 5 kJ/mol. Compare to Fig. 3. In this context, it is worth mentioning the relative differences for Hanatoxin dissociation constants in the closed state of the drk1 VSD, as reported by Swartz [34] and Li-Smerin [35]. While the Hanatoxin data measures the relative stability of O vs C states rather than the kinetics, their study found a 25-fold change upon mutating away the phenylalanine (F274 in drk1), which corresponds to a free energy stabilization of 8 kJ/mol for the open relative to the closed state. The same study reported 13-fold change for the preceding position (6.3 kJ/mol, I273 in drk1), 15-fold change three positions later (6.7 kJ/mol, E277 in drk1), and successively smaller changes for the arginines (R0 4.2 kJ/mol, R2 4 kJ/mol, R3 2.7 kJ/mol, R4 2.1 kJ/mol; R1 in Kv1.2 is a glutamine in drk1). This pattern combined with the limited effect on stability in O vs. C states observed here suggests these mutations primarily destabilize the closed state of the channel.