pH-dependent Inhibition of Voltage-gated H(super +) Currents in Rat Alveolar Epithelial Cells by Zn(super 2+) and Other Divalent Cations

Inhibition by polyalent 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 ZnCl(sub 2) reduced the H(super +) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(super +) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(super 2+) were inconsistent with classical voltage-dependent block in which Zn(super 2+) binds within the membrane voltage field. Instead, Zn(super 2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(super 2+) were strongly pH(sub o) dependent but were insensitive to pH(sub i), suggesting that protons and Zn(super 2+) compete for external sites on H(super +) channels. The apparent potency of Zn(super 2+) in slowing activation was approximately 10X greater at pH(sub o) 7 than at pH(sub o) 6, and approximately 100X greater at pH(sub o), 6 than at pH(sub o) 5. The pH(sub o) dependence suggests that Zn(super 2+), not ZnOH(super +), is the active species. Evidently, the Zn(super 2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(super 2+) binding. The external receptor bound H(super +) and Zn(super 2+) with pK(sub a) 6.2-6.6 and pK(sub M) 6.5, as described by several models. Zn(super 2+) effects on the proton chord conductance-voltage (g(sub H)-V) relationship indicated higher affinities, pK(sub a) 7 and pK(sub M) 8. CdCl(sub 2) had similar effects as ZnCl(sub 2) and competed with H(super +), but had lower affinity. Zn(super 2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(super +) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(super 2+) receptor may be the same modulatory protonation site(s) at which pH(sub o) regulates H(super +) channel gating.