The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage gated proton channel1, hHV1. This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2–4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (a) a titratable amino acid residue in the permeation pathway imparts proton selectivity1, 8–11, or (b) water molecules “frozen” in a narrow pore conduct protons while excluding other ions12. Here we identify Aspartate112 as a crucial component of the selectivity filter of hHV1. When a neutral amino acid replaced Asp112, the mutant channel lost proton specificity and became anion selective or did not conduct. Only the glutamate mutant remained proton specific. Mutation of the nearby Asp185 did not impair proton selectivity, suggesting that Asp112 plays a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp112, the mutant channel was still anion permeable. Evidently, the proton specificity of hHV1 requires an acidic group at the selectivity filter.
Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis, and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.
Fogel and Hastings first hypothesized the existence of voltagegated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH V 1: it is proton-specific and activated by depolarization, its g H -V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp 51 ) results in loss of proton-specific conduction. Indirect evidence suggests that kH V 1 is monomeric, unlike other proton channels. Furthermore, kH V 1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E H . This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.ion selectivity | ion channel | permeation | channel gating | action potential
The topological similarity of voltage-gated proton channels (HV1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether HV1s have a similar structure. We present the construction and validation of a homology model of the human HV1 (hHV1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHV1 by exploiting the homology of hHV1 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 hHV1 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 Asp112. Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHV1. Each Arg residue in the S4 helix of hHV1 was replaced by His to test accessibility using Zn2+ 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, Arg211, the third Arg residue in the S4 helix in hHV1, remains accessible to the internal solution and is located near the charge transfer center, Phe150.
Voltage-gated proton channels are strongly inhibited by Zn 2+ , which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn 2+ . We hypothesize that high-affinity Zn 2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn 2+ effects were weaker in monomeric channels. Mutation of His 193 and His 140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn 2+ only when at least one His was present in each monomer, suggesting that in wild-type (WT) H V 1, Zn 2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn 2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.
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