While the double mutation of R3 and D112 to serine (D112S-R3S) produced the largest disruption that we observed of ion selectivity, the charge swap (D112R-R3D) retained proton selectivity. Together, VX-770 price these observations suggest that D112 and R3 interact electrostatically to contribute to the selectivity filter of the channel, and that mutation of R3 alone or in combination with D112S

induces a voltage sensor that leaks cations other than protons (Figure 8B). The number of mutations required to create a pore in a VSD provides information on the length and shape of the pore’s most constricted site. The “omega pore” through the VSD of the Shaker K+ channel requires a single mutation of the first arginine R1 to a small side chain (Tombola et al., 2005), leading to its opening when S4 is in the “down state” at hyperpolarized potentials. However, it appears that Shaker actually requires a double gap (substitution of two arginines) and that the outermost position (three residues before Shaker’s R1) is naturally “missing” (i.e., is an alanine), while an omega pore can also be made in Shaker at other voltages by mutations at neighboring Androgen Receptor Antagonist pairs of arginines (Gamal El-Din et al., 2010). A double gap is also needed to create an omega pore through the

VSD in domain II of Nav1.2a, (Sokolov et al., 2005). However, in domain II of Nav1.4 channels, mutation of a single arginine (R2 or R3) is sufficient to make an omega current (Struyk et al., 2008, Sokolov et al., 2008 and Sokolov et al., 2010). We find that a single gap is sufficient for hHv1 to conduct Gu+, indicating that the pore of hHv1 is relatively short. As we observe here with hHv1, the Shaker omega pore is more permeable to Gu+ than to metal cations (Tombola et al., 2005). Moreover, Gu+ and protons have been found to be highly permeable through the VSD of domain II of Nav1.4 Na+ channel when Adenylyl cyclase a single arginine gap is made by substitution with glycine or histidine (Sokolov et al., 2010). Thus, the hHv1 VSD pore pathway shares with its counterparts from K+ and Na+ channels

a preference for the free ion that resembles the arginine side chain. A remarkable feature that appears to distinguish the omega pore of hHv1 from that of other channels is that arginine is uniquely able to select against Gu+, whereas other bulky or charged residues do not. Recent molecular dynamics simulations based on homology models built upon voltage-gated K+ channel crystal structures showed that water can occupy the core of the VSD of hHv1, but not of VSDs of tetrameric channels, suggesting that hHv1 may have evolved a specialized watery proton transfer pathway (Ramsey et al., 2010). Our findings are compatible with such a transfer pathway and with details of the homology model on which the simulations were based on, namely the close proximity of D112 to R3 in the activated state.