Rebuttal from Ashley L. Bennett and Ian Scott Ramsey

DeCoursey suggests that ‘frozen water’ may block cation flux through the central crevice of the Hv1 VS domain. However, individual water molecules are dynamic, and may enter or leave this site during MD simulations (Ramsey et al. 2010; Wood et al. 2012; Kulleperuma et al. 2013). Finite time‐averaged water occupancy at the constriction point (near D112/D^1.51, F150/F^2.50 and either R2/R208/R^4.50 or R3/R211/R^4.53, depending on the model) evidently results from transient hydrogen bonds between water and protein atoms that restrict water mobility (Ramsey et al. 2010, Wood et al. 2012; Kulleperuma et al. 2013). Consistent with our hypothesis that the ‘aqueous’ conductance (G _AQ) is mediated by an ensemble of interconvertible water–protein hydrogen bond structures, experimental evidence demonstrates that Hv1 is remarkably refractory to amino acid mutagenesis (Ramsey et al. 2010; Musset et al. 2011; Morgan et al. 2013; Randolph et al. 2016). The water–protein hydrogen bond network in Hv1 may also explain biophysical properties of PT that are distinct from ice or simple water wires (i.e. gramicidin A), as outlined by DeCoursey.

Experimental data indicate that maintaining charge at D112/D^1.51 and R3/R211/R^4.53 is necessary for high H⁺ selectivity (Berger & Isacoff, 2011; Musset et al. 2011), suggesting that the flux of mobile cations and anions through Hv1 is prevented by the fixed anion or cation at these sites. Neutralization of D112/D^1.51 during PT, as proposed by DeCoursey and colleagues (Dudev et al. 2015), would therefore also be expected to erode selectivity. A more parsimonious explanation is that waters mediate PT and create a pathway for the diffusion of mobile solution ions that is readily measurable only in mutant Hv1 channels.

Furthermore, D112V is the only mutation that is convincingly non‐functional (Musset et al. 2011; Morgan et al. 2013); other substitutions are tolerated and H⁺‐permeable (Musset et al. 2011). Replacement of Asp with the aliphatic Val could allow 112 V to interact with other nearby hydrophobic groups, potentially expelling waters from the central crevice and trapping the channel in a PT‐incompetent conformation. We accordingly find that the D112/D^1.51 side chain is nestled between I^2.46, L^2.47, V^3.53 and I^4.44 in a resting‐state Hv1 model structure (Randolph et al. 2016).

Lastly, we note that the guanidinium ion (Gu⁺) is structurally identical to the side chain of Arg residues that constitute gating charges in the S4 helix (Gonzalez et al. 2013). The observation that N214R (N4R) results in voltage‐dependent block of activated‐state G _AQ indicates that voltage‐dependent movement of S4 Arg side chains through the central crevice (i.e. ‘gating pore’) is sufficient to explain open–closed gating in Hv1 (Randolph et al. 2016), as proposed previously (Vargas et al. 2012; Gonzalez et al. 2013). In the Hv1 B model, the R3/R211/R^4.53 side chain is extracellular to D112/D^1.51, allowing G _AQ to remain open. If Gu⁺ fits into this crevice better than other similarly sized monovalent organic cations, it could permeate without breaking salt bridges.

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None declared.

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