Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hH(V)1

Part of the “signature sequence” that defines the voltage-gated proton channel (H(V)1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H(V)1 genes. Replacing Trp(207) in human H(V)1 (hH(V)1) 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, E(a), for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp(207) establishes the major energy barrier between closed and open hH(V)1. Cation–π interaction between Trp(207) and Arg(211) evidently latches the channel closed. Trp(207) mutants lost proton selectivity at pH(o) >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H(V)1 that is essential to its biological functions, was compromised. In the WT hH(V)1, ΔpH-dependent gating is shown to saturate above pH(i) or pH(o) 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 Trp(207) mutants, ΔpH-dependent gating saturated at lower pH(o) but not at lower pH(i). That Trp(207) mutation selectively alters pH(o) sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H(V)1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH(o) and pH(i) in H(V)1 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.