Resting state of the human proton channel dimer in a lipid bilayer

Abstract

The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage-dependent cation channels and enzymes. We set out to determine the structural properties of membrane-reconstituted human proton channel (hHv1) in its resting conformation using electron paramagnetic resonance spectroscopy together with biochemical and computational methods. We evaluated existing structural templates and generated a spectroscopically constrained model of the hHv1 dimer based on the Ci-VSD structure at resting state. Mapped accessibility data revealed deep water penetration through hHv1, suggesting a highly focused electric field, comprising two turns of helix along the fourth transmembrane segment. This region likely contains the H + selectivity filter and the conduction pore. Our 3D model offers plausible explanations for existing electrophysiological and biochemical data, offering an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement.V oltage-gated proton channels (hHv1) represent a remarkable evolutionary adaptation of canonical voltage sensing domain (VSD). In hHv1, both voltage-sensing and ion (H + ) conduction are carried out by a single domain, and undergo a global conformational rearrangement (1, 2). In humans, hHv1 is widely expressed and required for a variety of physiological processes (3), including optimal reactive oxygen species production by NADPH oxidase (4-6), B-cell proliferation and differentiation (7), and regulation of human sperm motility (8). hHv1 folds as an antiparallel four-helix bundle with its fourth transmembrane segment (S4) containing three putative gating charges. Just as in the VSDs from ion channels and enzymes, the positively charged S4 moves outwardly in response to a depolarization (9). Beyond sensing the transmembrane voltage, S4 reorientation in hHv1 participates in the formation of a protonselective permeation pathway responsible for the generation of the proton currents that underlie its multiple physiological functions (10, 11).Despite high sequence similarity and a common structural blueprint, VSDs from ion channels and enzymes display a wide range of electrophysiological properties; these include large variations in effective gating charge (z = ∼0.9 to ∼3.6 e − ) and midpoints of activation (V 1/2 = ∼−150 to ∼+150 mV). Existing VSD crystal structures (12-16) have provided key data on the arrangement and orientation of the transmembrane helices S1-S4, while at the same time revealed a considerable structural heterogeneity-particularly, the number and positions of both the gating charges and their compensating countercharges in relation to the hydrophobic "plug" or "gasket" electrically separating the intra-and extrace...

On the role of water density fluctuations in the inhibition of a proton channel

“…4 and SI Appendix, Results) can be defined on the basis of distances involving the signature residues R3 and F150 at the two constriction sites (34). Interestingly, such conformational heterogeneity is consistent with the electron paramagnetic resonance (EPR) experiments by Li et al (37), showing that Hv1 is significantly more flexible than other VSDs. We now describe in detail the conformational ensemble visited in the three A1 to A3 substates.…”

“…A number of homology models of Hv1 have been generated and validated (32)(33)(34)(35)(36)(37)(38); most of them were created using other VGICs as templates and the sequence of nonhuman orthologs. Here, we used the crystal structure of mouse Hv1 (31) as a basis to model all of the relevant conformational states of the human Hv1 monomer: resting, intermediate-resting, and activated states.…”

“…In both cases, water penetrates throughout the channel lumen. In particular, two major hydration crevices are located at the innermost and outermost ends of the channel (34,35,37), whereas a restricted area (from −6 to 6 Å along the z axis) is significantly less populated (Fig. 3).…”

“…These discoveries, and the ensuing potential therapeutic strategies, ignited extensive searches for effective Hv1 inhibitors; these efforts led to the identification of positively charged binders of the activated state (8,29,30). The lack of available experimental structures, only recently overcome by the discovery of the X-ray structure (31) of mouse Hv1, has motivated the generation of several comparative homology models using various VGICs as templates (32)(33)(34)(35)(36)(37)(38). Nonetheless, none of these studies focused on a systematic characterization of Hv1 as a pharmacological target using a structure-based approach for drug design.…”

Structural revelations of the human proton channel

“…Li et al (3) combine biochemical, computational, and electron paramagnetic resonance (EPR) spectroscopic approaches to shed light on structural aspects of the human proton channel, hH V 1. Their results advance the field in several key areas, culminating in a bold new model for gating.…”