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Voltage-gated proton channels open appropriately in myriad physiological situations because their gating is powerfully modulated by both pHo and pHi. Cherny et al. serendipitously identify a histidine at the inner end of the S3 helix that is required for the response to pHi., We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the voltage-gated proton channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1—changing His168 to Gln168, the corresponding residue in HtHV1—compromises the pHi dependence of gating in the human channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus. His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by

Voltage-gated proton currents were first discovered in snail neurons. Thomas et al. identify a snail proton channel gene that codes for a rapidly activating proton channel that differs from other proton channels, in particular by its low sensitivity to intracellular pH., Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo. Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance–voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi. This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).

A conserved tryptophan uniquely present in the voltage-sensing domain of voltage-gated proton channels is surprisingly crucial to four channel-defining properties., Part of the “signature sequence” that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation–π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1, ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.

The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H(V)1s have a similar structure. We present the construction and validation of a homology model of the human H(V)1 (hH(V)1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH(V)1 by exploiting the homology of hH(V)1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH(V)1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH(V)1. Each Arg residue in the S4 helix of hH(V)1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hH(V)1, remains accessible to the internal solution and is located near the charge transfer center, Phe(150).