With a single gene encoding H_V1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian H_V1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of H_V1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of H_V channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding H_V channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different H_V channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.

Gating of the voltage-gated proton channel H_V1 is strongly controlled by pH. There is evidence that this involves the sidechains of titratable amino acids that change their protonation state with changes of the pH. Despite experimental investigations to identify the amino acids involved in pH sensing only few progress has been made, including one histidine at the cytoplasmic side of the channel that is involved in sensing cellular pH. We have used constant pH molecular dynamics simulations in symmetrical and asymmetrical pH conditions across the membrane to investigate the pH- and ΔpH-dependent gating of the human H_V1 channel. Therefore, the pK_a of every titratable amino acids has been assessed in single simulations. Our simulations captured initial conformational changes between a deactivated and an activated state of the channel induced solely by changes of the pH. The pH-dependent gating is accompanied by an outward displacement of the three S4 voltage sensing arginines that moves the second arginine past the hydrophobic gasket (HG) which separates the inner and outer pores of the channel. H_V1 activation, when outer pH increases, involves amino acids at the extracellular entrance of the channel that extend the network of interactions from the external solution down to the HG. Whereas, amino acids at the cytoplasmic entrance of the channel are involved in activation, when inner pH decreases, and in a network of interactions that extend from the cytoplasm up to the HG.

Somatic mutations are common in cancer, with only a few driving the progression of the disease, while most are silent passengers. Some mutations may hinder or even reverse cancer progression. The voltage-gated proton channel (H_V1) plays a key role in cellular pH homeostasis and shows increased expression in several malignancies. Inhibiting H_V1 in cancer cells reduces invasion, migration, proton extrusion, and pH recovery, impacting tumor progression. Focusing on HVCN1, the gene coding for the human voltage-gated proton channel (hH_V1), 197 mutations were identified from three databases: 134 missense mutations, 51 sense mutations, and 12 introducing stop codons. These mutations cluster in two hotspots: the central region of the N-terminus and the region coding for the S4 transmembrane domain, which contains the channel’s voltage sensor. Five somatic mutations within the S4 segment (R205W, R208W, R208Q, G215E, and G215R) were selected for electrophysiological analysis and MD simulations. The findings reveal that while all mutants remain proton-selective, they all exhibit reduced effective charge displacement and proton conduction. The mutations differentially affect hH_V1 kinetics, with the most pronounced effects observed in the two Arg-to-Trp substitutions. Mutation of the first voltage-sensing arginine (R1) to tryptophan (R205W) causes proton leakage in the closed state, accelerates channel activation, and diminishes the voltage dependence of gating. Except for R205W, the mutations promote the deactivated channel configuration. Altogether, these data are consistent with impairment of hH_V1 function by mutations in the S4 transmembrane segment, potentially affecting pH homeostasis of tumor cells.

Many protein functions are coupled to proton transfer, yet the underlying mechanisms remain extremely difficult to resolve by experiment or conventional molecular dynamics (MD) simulations. The human voltage-gated proton channel H _V 1 (hH _V 1) plays key roles in human physiology, but how voltage and pH together drive its activation remains unresolved. Experimental structures of hH _V 1 are limited to the X-ray structure of a mouse chimera and NMR models of a truncated hH _V 1, both representing an intermediate resting state. Here we determine the activation pathway of hH _V 1, together with structural models of its resting and activated states, using an approach that integrates all-atom continuous constant-pH MD (CpHMD), weighted-ensemble (WE) path sampling, and AlphaFold modeling. Starting from an AlphaFold model selected for consistency with resting-state experimental data, over 100 µ s of simulations reveal a mechanism in which transmembrane voltage, assisted by proton titration, drives an outward displacement of the transmembrane segment S4 accompanied by a one-click reorganization of countercharge pairings. These events are enabled by a delicately balanced electrostatic landscape and pH-modulated breathing motions in the resting state that prime the channel for protonation-state switches upon voltage sensing, a principle that may underlie other proton-coupled voltage-gating processes. The simulations also uncover lipid-binding sites at which POPG selectively stabilizes the S4-up conformation, whereas POPE can stabilize either state. These predictions are validated against extensive electrophysiology, mutagenesis data, and new smFRET experiments, which support the one-click S4 displacement and the lipid effects. WE-CpHMD, implemented in the AMBER package, is applicable to other proton-coupled processes that remain elusive.