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Original Article

^a From the Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612
Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612.312-942-8711

tdecours{at}rush.edu

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 10x greater at pH_o 7 than at pH_o 6, and 100x 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.

Key Words: metal binding constants • cadmium • pH • hydrogen ion • ion channels

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