pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) 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 ZnCl(2) 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 Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) 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 Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.
Cherny
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Department of Molecular Biophysics, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
DeCoursey
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D000465:Algorithms; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002021:Buffers; D002413:Cations, Divalent / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D015032:Zinc / Q000494:pharmacology*
D002021:Buffers; D002413:Cations, Divalent; D007473:Ion Channels; D006859:Hydrogen; D015032:Zinc