Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

• Published in: The Journal of biological chemistry 2013-06, Vol.288 (25), p.17968-17975
• Main Authors: Fujiwara, Yuichiro, Takeshita, Kohei, Nakagawa, Atsushi, Okamura, Yasushi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biosensors; Blotting, Western; Circular Dichroism; Coiled Coil; Crystallography, X-Ray; Disulfides - chemistry; Disulfides - metabolism; HEK293 Cells; Humans; Ion Channel Biophysics; Ion Channel Structure-Function; Ion Channels; Ion Channels - chemistry; Ion Channels - genetics; Ion Channels - physiology; Macrophages - metabolism; Membrane Potentials; Mice; Models, Molecular; Molecular Biophysics; Molecular Sequence Data; Mutation; Oxidation-Reduction; Patch-Clamp Techniques; Phagosomes - metabolism; Protein Multimerization; Protein Structure; Protein Structure, Quaternary; Protein Structure, Tertiary; Proton Channel; Proton Transport; Redox; Sequence Homology, Amino Acid; Sulfhydryl Compounds - chemistry; Sulfhydryl Compounds - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.459024

Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H+ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome. A pair of Cys residues is present in the coiled-coil assembly domain in the Hv channel dimer. An intersubunit disulfide bond forms in a redox-dependent manner and stabilizes the dimeric assembly. The Hv channel has a redox sensor in the cytoplasmic region. Hv channels expressed in phagocytes may sense the redox condition in production of reactive oxygen species for repelling bacteria.