Ph-Dependent Inhibition of Voltage-Gated H⁺ Currents in Rat Alveolar Epithelial Cells by Zn²⁺ and Other Divalent Cations

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl₂ reduced the H⁺ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H⁺ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn²⁺ were inconsistent with classical voltage-dependent block in which Zn²⁺ binds within the membrane voltage field. Instead, Zn²⁺ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn²⁺ were strongly pH_o dependent but were insensitive to pH_i, suggesting that protons and Zn²⁺ compete for external sites on H⁺ channels. The apparent potency of Zn²⁺ in slowing activation was ∼10× greater at pH_o 7 than at pH_o 6, and ∼100× greater at pH_o 6 than at pH_o 5. The pH_o dependence suggests that Zn²⁺, not ZnOH⁺, is the active species. Evidently, the Zn²⁺ receptor is formed by multiple groups, protonation of any of which inhibits Zn²⁺ binding. The external receptor bound H⁺ and Zn²⁺ with pK_a 6.2–6.6 and pK_M 6.5, as described by several models. Zn²⁺ effects on the proton chord conductance–voltage (g_H–V) relationship indicated higher affinities, pK_a 7 and pK_M 8. CdCl₂ had similar effects as ZnCl₂ and competed with H⁺, but had lower affinity. Zn²⁺ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H⁺ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn²⁺ receptor may be the same modulatory protonation site(s) at which pH_o regulates H⁺ channel gating.