title: Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1.
abstract: Part of the "signature sequence" that defines the voltage-gated proton channel (H(V1)) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H(V1) genes. Replacing Trp207 in human HV1 (hH(V1)) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hH(V1). Cation-π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H(V1) that is essential to its biological functions, was compromised. In the WT hH(V1), ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H(V1) from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in H(V1) of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Cherny
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612.
authors.forename: Deri
authors.initials: D
authors.lastname: Morgan
authors.affiliation: Institute of Complex Systems 4 Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Musset
authors.affiliation: Institute of Complex Systems 4 Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich, Germany.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Chaves
authors.affiliation: Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA 30144.
authors.forename: Susan M E
authors.initials: SM
authors.lastname: Smith
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612 tdecours@rush.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: DeCoursey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: C510235:HVCN1 protein, human; D007473:Ion Channels; D014364:Tryptophan