Selectivity Mechanism of the Voltage-gated Proton Channel, HV1

Abstract

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp-Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O(+) protonated the SF, breaking the Asp-Arg linkage and opening the conduction pathway, whereas Na(+) or Cl(-) was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp-Lys SF behaved like the Asp-Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH(0)-H2O(0)-Arg(+) interactions with hydronium but unfavorable Asp(-)-X(-)/X(+)-Arg(+) interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling.

Figure 1. Binding of H₃O⁺ to the Asp–Arg SF.

Fully optimized B3-LYP/6-31+G(3d,p) structures of (a) ion-free Asp^––Arg⁺ SF, (b) Asp⁰–Arg⁰ SF, (c) initial configurations of the SF-H₃O⁺ complex and (d) final configuration of the SF–H₃O⁺ complex, AspH⁰–H₂O–Arg⁺ with H in grey, C in green, N in blue and O in red. A dashed line denotes a hydrogen bond, which is defined by a donor–acceptor distance ≤3.5 Å and a H–acceptor distance ≤2.5 Å. The reaction between SF and H₃O⁺ is depicted in (e) with free energies given in kcal/mol; ΔG¹ is the binding free energy in the gas phase, whereas ΔG⁴ and ΔG³⁰ are the corresponding free energies in the SF characterized by an effective dielectric constant of 4 and 30, respectively.

Figure 2. Binding of Cl^– and Na⁺ to Asp–Arg SF.

Ball and stick diagrams of the initial (left) and final (right) structures of SF complexes with (a) Cl^– and (b) Na⁺.

Figure 3. Free energies (in kcal/mol) for replacing H₂O bound in Asp–Arg SF with H₃O⁺.

See Fig. 1 legend.

Figure 4. (a) Free energies (in kcal/mol) for binding of H₃O⁺ to Lys mutant SF.

Ball and stick diagrams of the initial (left) and final (right) structures of Arg → Lys mutant SF complexes with Cl^– (b) and Na⁺ (c). See Fig. 1 legend.

Figure 5. The Lys208 mutant is proton selective.

Measured values of V_rev at ΔpH –1.0, 0, or 1.0 (mean ± SEM, n = 3, 9, or 6, respectively), with pH_o ranging 5.5 to 7.0 and pH_i ranging 5.5 to 8.0. The linear regression slope was 53.3 mV/unit ΔpH, compared with the Nernst value of 58.4 mV. Inset: Proton currents in an inside out patch during pulses applied in 5 mV increments (left) indicate reversal between 0 and 5 mV (the conductance activated negative to V_rev) at pH_i 7.0, with pH_o 7.0 (in the pipette). Tail currents in the same patch at pH_i 6.0 indicate reversal at –58 mV. Both values are near the Nernst predictions of 0 mV and –58.4 mV.

Figure 6. Binding of Cl^– and/or OH^– to H₃O⁺-bound mutant SFs.

B3LYP/6-31+G(3d,p) fully optimized structures of H₃O⁺–SF, Cl^––SF and OH^––SF complexes, and free energies (in kcal/mol) for (a) [SF(Ala-Arg⁺)-H₃O⁺] + Cl^– → [SF(Ala-Arg⁺)-Cl^–] + H₃O⁺, (b) [SF(Ala-Arg⁺)-H₃O⁺] + OH^– → [SF(Ala-Arg⁺)-OH^–] + H₃O⁺, and (c) [SF(His-Arg⁺)-H₃O⁺] + Cl^– → [SF(His-Arg⁺)-Cl^–] + H₃O⁺. ΔG¹ is the ion exchange free energy in the gas phase, whereas ΔG⁴ and ΔG³⁰ are the corresponding free energies in the SF characterized by an effective dielectric constant of 4 and 30, respectively. If the resulting free energy is negative, the pore is Cl^– or OH^–-selective, but if it is positive, the pore is proton-selective.

Figure 7. Schematic cartoon of the proposed proton selectivity mechanism by the H_V1 SF.

Negatively charged Asp is red, neutral AspH⁰ and H₂O⁰ are green, whereas positively charged H₃O⁺ and Arg are light and dark blue, respectively. The dashed lines denote hydrogen bonds or salt bridges that occlude the SF pore. When H₃O⁺ approaches the SF (left), it breaks the hydrogen bonds and protonates the SF, resulting in neutral H₂O bridging AspH⁰ and Arg⁺ (middle). Transfer of a proton from the SF to H₂O completes the conduction cycle (right).

Figure 8. A critical Asp-Arg pair in F₁-F_o ATPase shares similar geometry to that in H_V1.

Based on a homology model of H_V1 in the open state and the crystal structure of F₁-F_o ATPase (PDB ID 1C17), Asp112 in H_V1 was superimposed onto Asp61 of F₁-F_o subunit c using Chimera, which minimizes the root-mean-square deviations of superimposed atoms. This resulted in Arg208 of H_V1 occupying a similar position to Arg210 of F₁-F_o subunit a, which is known to participate in proton translocation.