In contrast to I287, increasing the hydrophobicity of V363 stabilizes the resting versus active VS conformation.

Hence, the endogenous Thr present at the homologous position in the VS of Nav DI–DIII destabilizes the resting state relative to the activated state, consequently reducing the energy barrier underlying VS activation (Figure 4E). This mechanism agrees well with previous works showing that the replacement of the native residues intercalated between the Shaker S4 Arg by less hydrophobic amino LY294002 supplier acids destabilizes the resting versus the depolarized VS conformation (Xu et al., 2010). Several molecular dynamics simulations of the resting conformation of the Kv1.2 voltage sensor show that the side chain of the residue homologous to V363 points toward the lipid

bilayer (Delemotte et al., 2011, Henrion et al., 2012, Jensen et al., 2010, Khalili-Araghi et al., 2010, Lacroix et al., 2012 and Vargas et al., 2011). In the VS resting state, this residue is therefore probably surrounded by the hydrophobic environment of the lipid bilayer and completely buried from the solvent (Figure 4F). Hence, this VS conformation will be energetically more stable when this residue bears a hydrophobic side chain and conversely will be less stable when this side chain is made more hydrophilic (Figure 4E). Interestingly, the presence MI-773 purchase of two hydrophilic residues in S4-DIII, one after K1 and one after R2 (Figure 2A and Figure S2), may constitute the molecular basis to account for the earlier activation-onset of domain III during sodium channel activation (Chanda and Bezanilla, 2002 and Gosselin-Badaroudine et al., 2012). The mutation V363I produces the largest positive Q-V

shift. Interestingly, the homologous mutation T220I in S4-DI of Nav1.5, a cardiac-specific Nav channel, is associated with early development of dilated cardiomyopathy (Olson et al., 2005). Figure S5 shows that the T220I mutation produces a positive shift of approximately +10 mV for both the channel’s availability and open probability, in agreement with the V363I phenotype. The proposed mechanism for the S4 speed-control site was further tested by conducting similar experiments Vasopressin Receptor in the unrelated VS from the Ciona Intestinalis voltage-sensitive phosphatase (Ci-VSP). Figure 5 shows that decreasing the hydrophobicity of the side chain at position L224, homologous to V363 in Shaker, negatively shifted the Q-V curve and accelerated the activation kinetics but did not significantly alter deactivation kinetics. Thus, similar mutations of this residue produce similar effects in two evolutionary-distant VSs. From the point-of-view of evolution, it is tempting to hypothesize that the rapid VSs that characterize Nav channels were designed by natural selection during the development of nervous systems.