Structural revelations of the human proton channel.

The voltage-gated proton channel, HV1, is notoriously unique among ion channels (1), and plays key roles in the health and disease of diverse tissues and species (2). 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, hHV1. Their results advance the field in several key areas, culminating in a bold new model for gating. The voltage-sensing domain (VSD) is the part of voltage-gated ion channels that senses the electrical potential across the cell membrane where the channel resides. Most such channels open upon membrane depolarization, which is accomplished mainly by cationic amino acids located in the fourth transmembrane helical segment (S4) moving outward when the inside of the cell is made more positive. This movement is transduced from the VSD (S1–S4) to the pore region (S5–S6), opening a conduction pathway. In addition to biologically important voltagegated K, Na, Ca, and H channels, other classes of membrane proteins with VSDs exist that are not channels at all. One example is a voltage-sensing phosphatase (VSP), an enzyme whose activity is regulated by membrane potential. In a landmark study in 2014 (4), the Li–Perozo group reported crystal structures for CiVSP in both “down” and “up” conformations, the first VSD-containing molecule to have structures determined in both states. Voltage-gated ion channels are closed at negative voltages, and open upon depolarization as the S4 helix moves “up” through the membrane electrical field. Because the VSP is not a channel, “closed” and “open” become “down” and “up.” The gene for hHV1 was identified only in 2006 (5). To the astonishment of everyone, the gene product bore a striking resemblance to the VSD of other voltage-gated ion channels, so much so that the simultaneously identified mouse (mHV1) and Ciona intestinalis (CiHV1) gene products were dubbed VSOP or “voltage sensor-only protein” (6). HV1 has only four membrane-spanning helices (S1– S4); these sense voltage but also contain the proton conduction pathway (7). Crystal structures are great, up to a point. They provide tremendously detailed information about molecules, but they have limitations that are sometimes overlooked. They tell us about structure, but only the structure of whatever exists in the crystal. Proteins have many conformations, and one must determine or guess which one was captured during crystallization, and hope it is a native conformation and not a broken one. Forming crystals of membrane proteins is challenging, and often the protein is modified to facilitate crystallization. Ligands or chaperone-like proteins are included, parts of the molecule are truncated, chimerae are produced: whatever it takes to get a good crystal. HV1 has not been successfully crystallized in its entirety. First came crystal structures of the C terminus alone that lacked the entire transmembrane region (8, 9). Then in 2014, the first exciting glimpse of HV1 appeared. Well, not quite HV1, but a chimera of the mouse proton channel, mHV1, with the C terminus replaced by a leucine zipper transcriptional activator GCN4 from Saccharomyces cerevisiae, and with the cytoplasmic ends of S2–S3 replaced by the corresponding section of CiVSP. Nevertheless, this three-species chimera functions as a proton channel and thus retains essential features. A protein in a crystal senses no membrane potential, and is assumed to be in a state occupied at 0 mV. This means HV1 is closed, although with no pH gradient, the channel begins to open within 20 mV (10). HV1 exhibits complex gating kinetics (10–12), suggesting it has multiple closed states. Because 0 mV is close to the “threshold” voltage where channels first open, the crystal may have captured a shallow closed state. EPR provides useful information not available from other approaches, but has its own limitations. Significantly, the protein could be studied in situ in its native environment, a lipid bilayer, in contrast to a crystal in which interactions with the membrane are lost. The greatest limitation may be the necessity to introduce a bulky spin label whose presence inside the protein may perturb the native structure at least locally. Li et al. (3) replaced the lone native cysteine (Cys) of hHV1 and then introduced Cys at each of 149 locations encompassing the entire VSD. A spin label was attached to each construct and the molecules reconstituted into liposomes. Both the full-length 273-amino acid protein and a VSD-only construct were purified and shown to mediate proton conduction in liposomes; VSD-only constructs were used in all EPR measurements. Three parameters obtained from EPR are mobility, O2 accessibility (which indicates proximity to lipid), and NiEDDA (Ni ethylenediaminediacetic acid) accessibility, which reports aqueous exposure. Both mobility and O2 accessibility are larger for hHV1 than for other VSD-containing molecules, revealing a dynamic molecule, deficient in tertiary contacts,