Mechanism of voltage-dependent gating in proton channels

Protons in open voltage-gated proton channels diffuse down the electrochemical gradient in a passive manner. The high turnover rate of 10⁵ H⁺ s^-1 of this process defines them as real channels instead of being a carrier protein (DeCoursey, 2003, 2017). Mainly, depolarization of the cell membrane from -70 mV resting potential to 40 mV induces a conformational change in the VSD, activating the channeling of protons. Thereby, HV1 gating is dependent on ∆pH and on the membrane polarization (DeCoursey, 2015). A special role is described for dinoflagellates where the electrochemical driving force is inward upon membrane depolarization contrary to the usual outward proton flux (Smith et al., 2011). Consequently, these channels were thought to have different functions.

The mechanism of proton flux through voltage-gated proton channels is controversially discussed (Bennett & Ramsey, 2017; DeCoursey, 2017). Based on different experimental data two mechanisms were postulated.

INTRODUCTION

First, protons can diffuse from the intracellular to the extracellular matrix by a Grotthus-type mechanism (Agmon, 1995). As described for the ion channel gramicidin A, the pore is filled with water where protons are transported via hopping along the water wire without direct involvement of amino acid sidechains (Myers & Haydon, 1972; Ramsey et al., 2010; Wood et al., 2012; Pupo et al., 2014; Randolph et al., 2016). Evidence is given by atomistic and homology models as well as molecular dynamic (MD) simulations where water filled crevices are detected (Ramsey et al., 2010; Wood et al., 2012; Pupo et al., 2014; van Keulen et al., 2017). Additionally, experts argue that more than 50 amino acid exchanges in the central pore of the HV1 channel still result in measurable currents, which is an indication that no amino acid side chains are involved in channeling processes (Bennett & Ramsey, 2017;

Ramsey et al., 2010; Randolph et al., 2016).

In contrast, the second hypothesis is based on the participation of amino acid side chains in proton transport in form of a hydrogen bonded chain mechanism (DeCoursey & Cherny, 1994, 1997; DeCoursey, 1998; DeCoursey & Cherny, 1998; DeCoursey, 2003). Here, the carboxyl group of a highly conserved aspartate in helix S1 (D112 in hHV1, potentially D69 in DrVSD) is protonated and deprotonated, acting as a proton shuttle (Musset et al., 2011;

Morgan et al., 2013; Dudev et al., 2015). The theory is based on different experimental results. First, the HV1 channel is highly selective for protons. A mutation of Asp112 (in hHV1) to a neutral amino acid converts the selectivity of the channel to anions (Musset et al., 2011). In a reduced quantum mechanical model of the HV1 selectivity filter, a hydrogen bond network between this Asp, neutral water and Arg side chains was observed, which occludes other ions from entering the pore (Dudev et al., 2015). Second, the observed isotope effect is contrary to the effect observed for protons in a water-filled gramicidin A channel and for protons in bulk solution (DeCoursey & Cherny, 1997). Furthermore, HV1 has a much higher temperature-dependence than observed for other ion channels or for protons in solution (DeCoursey & Cherny, 1998; Kuno et al., 2009), which can be explained by e.g. rotational processes of protonated side chains like described for the M2 influenza A virus proton channel (Lin & Schroeder, 2001; Hu et al., 2010). Fourth, kinetic measurements of transport rates in the HV1 gave evidence for an involved generic hydrogen bonded chain when compared with literature data (Nagle & Morowitz, 1978; DeCoursey, 2017).

INTRODUCTION

In addition to the two hypotheses of proton channeling, the inhibition of VSDs is controversially discussed too. HV1 channels coordinate Zn²⁺ by two histidine residues in the crystal structure (Mahaut-Smith, 1989; Takeshita et al., 2014). Nevertheless, homology models could not support this thesis completely (Musset et al., 2010c). Furthermore, voltage-gated proton channels in coccolithophores miss any His residues, but still show an inhibition by Zn²⁺ (Taylor et al., 2011). In sum, so far it is unclear which details are responsible for the HV1 channel inhibition by polyvalent cations.

Another inhibition by guanidine derivatives was intensively studied by mutational experiments and MD simulations, but no holo-structure is reported so far (Hong et al., 2013;

Hong et al., 2014a; Hong et al., 2015; Gianti et al., 2016). Nevertheless, potential residues in hHV1 are described, which are involved in intracellular e.g. 2-guanidinobenzimidazole (2GBI) binding with a KD of 38 µM (Hong et al., 2013; Hong et al., 2014a; Gianti et al., 2016).

Computational docking studies of hHV1 with 2GBI revealed a benzimidazole ring stabilization by the aa V109, L108, I146, I105, V178, F150, D112 and R211 (Gianti et al., 2016).

Furthermore, F182 is described to stabilize the guanidine moiety. Docking experiments were performed with an active model of hHV1 based on the mouse HV1 crystal structure.

Transferring the data to my homology models reveals slightly different results (Figure 5).

Figure 5: PyMOL-based 2GBI docking in modeled VSD structures. Structures of hHV1 (green) and DrVSD (orange) were modeled using SWISS-MODEL and the open CiVSD structure as template (Figure 2, Figure 4). The four transmembrane helices (S1-S4) as well as N- and C-terminal parts are displayed. Published residues involved in inhibitor binding (Gianti et al., 2016; Hong et al., 2014a) are shown as stick representations colored by element (O-red, N-blue). 2GBI is shown in a pink stick representation colored by element. The dotted lines represent a zoom of all atoms 6 Å within the 2GBI selection to highlight possible interaction partners.

INTRODUCTION

Interestingly, my applied model could not show any interaction of 2GBI with residues F182 and R211 in parallel although the other contacts could be identified. This demonstrates again the difficulties in detailed analysis of the binding and channeling mechanism.

Nevertheless, the binding region of 2GBI deep inside the channel promotes the theory of active-state inhibitor binding (Hong et al., 2013; Hong et al., 2014a; Hong et al., 2015).

Furthermore, the remarkable role of residue F150 in inhibitor binding can be shown, as the inhibitor has to pass this barrier to be able to bind to the channel. For example, mutational studies revealed an increased inhibition when F150 was exchanged to alanine (Hong et al., 2013; Hong et al., 2014a; Gianti et al., 2016).

In summary, there is an ongoing discussion about the proton channeling mechanism, the binding site for polyvalent cations and for guanidine derivatives in VSDs. However, a general mechanistic description for the proton flux through a voltage-gated proton channel is missing but of importance especially for future drug developments.