Hydrophobic plug functions as a gate in voltage-gated proton channels

Abstract

Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.voltage gating | mutagenesis cycle | molecular dynamics | VSOP | blocker T he best-studied function of voltage-gated proton (Hv1) channels is in the immune system, where the activity of Hv1 channels has been shown to play a key role in charge compensation for the electron extrusion by NADPH oxidase during the respiratory burst in phagocytes (1, 2). In addition, this channel is also found in many other cell types including neurons, sperm, and lung airway epithelia cells, where it has been implicated in acid extrusion, male fertility, and the pathology of asthma, respectively (3, 4). During stroke, the activity of Hv1 channels exacerbates neuronal death (5). In 2006, two independent groups identified the genes coding for the Hv1 channel in humans (hHv1) (6) and mice and Ciona intestinalis (voltage-sensing only protein; we call it "Ci-Hv1" in this paper) (7). The sequences of Hv1/(VSOP) are homologous to the sequences of the voltagesensing domain (VSD) of other voltage-gated ion channels, such as voltage-gated sodium, potassium, and calcium channels (Nav, Kv, and Cav channels), and the voltage sensor-containing phosphatase VSP (6, 7). Hydropathy analysis of the Hv1 sequence suggests the existence of four transmembrane (TM) segments (Fig. 1A), designated S1 to S4, similar to the four TM segments of the VSD of Kv channels (Fig. S1). However, Hv1 channels lack the last two TM segments of Kv channels that make up t...

Flagellar ion channels of sperm: similarities and differences between species

“…While Hv1 is ubiquitously expressed and plays an important role in a number of physiological processes such as the innate immune system, apoptosis, and cancer metastasis, the presence of Hv1 in human spermatozoa was intriguing. Hv1 is not a true ion channel, but rather a strange mix between a transporter and an ion channel without the pore, that provides rapid movement of protons across a lipid bilayer via a voltage-gated mechanism [116][117][118].…”

“…Unlike traditional voltage-gated ion channel, Hv1 does not possess a pore domain. Several models for the proton conductance pathway have been proposed [116,117,126]. The "water wire" model [117] proposes the movement of protons via Grotthus mechanism from the intracellular water vestibules to the extracellular vestibule formed by the transmembrane (TM) portions of the channel.…”

“…Two vestibules are connected by a narrow bottleneck [117] where proton selectivity occurs via a highly conserved and unique aspartate moiety (Asp112 in human Hv1, [127]). Another model [116] suggests that Hv1 closed state favors electrostatic interactions between hydrophobic residues of TM segments and pull them together to form a hydrophobic plug. This hydrophobic layer is formed at the center of the voltage-sensing domain and prevents proton permeation.…”

“…This hydrophobic layer is formed at the center of the voltage-sensing domain and prevents proton permeation. The latter occurs once the plug is removed by a voltage-dependent rearrangement of TMs and is followed by insertion of the protonatable residues in the center of the channel [116]. The recent crystal structure of the Hv1 in resting state sheds [118] light on the possible mechanism of H + permeation, suggesting that a combination of two hydrophobic plugs and a protonatable aspartate is required for proton movement.…”

Inhibitory mechanism of peptides with a repeating hydrophobic and hydrophilic residue pattern on interleukin-10

“…50 Recently, it has been found that Arg directly associates with the opening and closing of voltage-gated proton channels, which play important roles in various physiological processes, including the innate immune response. 51 Thus, promoting the effect by having more Arg may be counteracted by simultaneously introducing structure instability.…”