Voltage and pH sensing by the voltage-gated proton channel, HV1

Abstract

Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high pK_a) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their pK_a needs to be within the operational pH range. We propose a 'counter-charge' model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups.

Keywords: ion channels; pH; proton conduction; proton transport; voltage gating.

Figure 1.

Architectural features of voltage-gated K⁺ channels, H_V1 channels and voltage-sensing phosphatases (VSP). The top row shows monomeric subunits of the complete molecule in the lower row. K⁺ channels are homotetramers with six transmembrane helices per monomer. Segments S1–S4 form the voltage-sensing domain (VSD) and S5–S6 form the conduction pathway. In the complete assembled channel (below), four VSDs (each comprising S1–S4) surround a single central pore through which K⁺ permeates. Dashed lines indicate central aqueous regions inside each VSD. H_V1 resembles an isolated VSD with only four TM segments and no explicit pore domain [5,6], but functions without accessory proteins [51]. It forms a dimer, largely due to coiled-coil interaction in the C terminus, but each protomer has its own conduction pathway [–54]. Phosphorylation of Thr²⁹ in the N terminus [8,55] greatly enhances H_V1 activity [56], especially in phagocytes [57]. The VSP lacks conduction altogether, but senses voltage and modulates phosphatatse activity accordingly [58,59]. Reprinted with permission from DeCoursey [60] (Copyright © 2010 American Physiological Society).

Figure 2.

Cooperative gating of H_V1. The H_V1 dimer behaves as expected of a classical Hodgkin–Huxley n² system. The WT channel in most species is a dimer in which both protomers must undergo a conformational change before either conducts. This manifests as sigmoidal activation kinetics in hH_V1 (a). Truncation of the C terminus eliminates coiled-coil interaction and results in each monomer apparently functioning independently. In monomeric constructs (b), activation is exponential and is five to seven times faster than in the dimer [52,83,84]. Lines show single exponential fits; both currents were recorded at +50 mV at a symmetrical pH of 7.5. In (c), the red trace is the fluorescence signal from a tag attached to S4 in dimeric CiH_V1, showing the exponential time course of its movement. The black trace shows the current, with its sigmoid turn-on. The green trace is the square of the red fluorescence signal, matching the current in the classical Hodgkin–Huxley manner, in which both protomers must activate before current is observed. (a,b) Reprinted with permission from Musset et al. [83] (Copyright © 2010 The Physiological Society) and (c) Reprinted with permission from Gonzalez et al. [85] (Copyright © 2010 Nature Publishing Group).

Figure 3.

Side view of the open human H_V1 channel, with the external end up. Transmembrane helices are colour-coded: S1 = red, S2 = yellow, S4 = blue and S3 is shown as lines to be unobtrusive. Key amino acids are labelled and shown with side chains as sticks. Asp¹¹² is crucial for selectivity; Phe¹⁵⁰ demarcates inner and outer aqueous vestibules and the three Arg in S4 sense voltage. Figure is based on the model of Li et al. [91]. Note that Asp¹¹² interacts with Asp²⁰⁸ [94], and Arg²¹¹ is below Phe¹⁵⁰, and thus is exposed to the inner vestibule. Drawn with PyMol.

Figure 4.

The ΔpH dependence of gating ensures that, in most species, H_V1 channels open only when doing so will result in acid extrusion. Each symbol or letter indicates a different cell type or species, defined in [68]. The blue line shows the relationship that results in H⁺ channels in almost all cells opening at their threshold voltage, V_threshold, at voltages positive to the Nernst potential (or the experimentally measured reversal potential, V_rev), shown as the red dashed line. At more positive voltages (above the blue line) more channels would open, extruding more protons. Reprinted with permission from DeCoursey & Hosler [69], with modification (Copyright © 2014 Rockefeller University Press).

Figure 5.

A four-state ‘butterfly’ model that explains the main features of ΔpH-dependent gating is shown with three possible physical representations. Channel opening occurs from left to right, with state 4 the only conducting state. Top row shows two ‘wings’ that cross the membrane exposing the sites to the opposite solution. The middle row depicts equivalent but distinct internal and external sites, of which only those on one side are accessible. The bottom row shows sites within the pore whose accessibility switches due to a subtle conformational change. Protonation from the external solution stabilizes the deepest closed (non-conducting) configuration (state 1). Deprotonation (state 1 → 2) is required before a conformational change switches the accessibility of the titratable groups to face inwardly (state 2 → 3). Finally, protonation from the inside (state 3 → 4) stabilizes the open channel (state 4). Because no single amino acid substitution abolishes ΔpH dependence [92], multiple groups are probably involved. Reprinted with permission from Cherny et al. [89] (Copyright © 1995 Rockefeller University Press).

Figure 6.

Cartoon illustrating the ‘counter-charge model’ for ΔpH-dependent gating of H_V1. The premise is that both open and closed states of the channel are stabilized by interhelical electrostatic interactions. The main charged groups on the S4 helix, which is thought to move outwards during channel opening, are three Arg (blue). The S1–S3 segments have a number of acidic groups. Internal protons (low pH_i) will tend to protonate acidic groups (a), preventing them from engaging in electrostatic interactions, thereby destabilizing the closed state and promoting opening. Conversely, external protons (low pH_o) will protonate externally accessible acidic groups, destabilizing the open state and promoting channel closing. Positions of groups are highly schematic! The hydrophobic gasket that demarcates internal and external accessibility is depicted as a highly conserved Phe¹⁵⁰ [143].