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pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Cherny VV, DeCoursey TE - J. Gen. Physiol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.

ABSTRACT
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.

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Mentions: The following buffers were used near their negative logarithm of the acid dissociation constant (pKa) (at 20°C) for measurements at the following pH: pH 5.0, Homopipes (homopiperazine-N,N ′-bis-2-(ethanesulfonic acid), pKa 4.61); pH 5.5–6.0 Mes (pKa 6.15); pH 6.5 BisTris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pKa 6.50); pH 7.0 PIPES (pKa 6.80); pH 7.5–8.0 HEPES (pKa 7.55). Buffers were purchased from Sigma Chemical Co., except for Homopipes (Research Organics). Buffers such as Tricine and BES that reportedly complex strongly with transition metals (Good et al. 1966) were avoided. We could not find information in the literature on the Zn2+ or Cd2+ binding properties of the buffers used. Therefore, we measured the binding constants for a number of buffers, according to the method described by Good et al. 1966. This consisted of titrating the buffer alone, and then together with an equimolar amount of the metal salt (usually 10 mmol in a 100-ml vol). The binding constant was calculated from the relationship (): 1\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K{\mathrm{^{\prime}}}_{{\mathrm{M}}}=\frac{2 \left \left(\displaystyle\frac{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }{K_{{\mathrm{a}}}}-1\right) \right }{ \left \left[{\mathrm{B}}\right] \right \left \left(\displaystyle\frac{K_{{\mathrm{a}}}}{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }+1\right) \right }{\mathrm{,}}\end{equation*}\end{document} where K ′M is the metal binding constant, Ka is the proton dissociation constant defined in Fig. 3 (−pKa value), [H+M] is the H+ concentration at the midpoint of the titration curve in the presence of the metal being tested and [B] is the total buffer concentration. The higher the affinity of the buffer for metal, the greater the shift in the titration curve. Table gives the results.


pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Cherny VV, DeCoursey TE - J. Gen. Physiol. (1999)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230650&req=5

Mentions: The following buffers were used near their negative logarithm of the acid dissociation constant (pKa) (at 20°C) for measurements at the following pH: pH 5.0, Homopipes (homopiperazine-N,N ′-bis-2-(ethanesulfonic acid), pKa 4.61); pH 5.5–6.0 Mes (pKa 6.15); pH 6.5 BisTris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pKa 6.50); pH 7.0 PIPES (pKa 6.80); pH 7.5–8.0 HEPES (pKa 7.55). Buffers were purchased from Sigma Chemical Co., except for Homopipes (Research Organics). Buffers such as Tricine and BES that reportedly complex strongly with transition metals (Good et al. 1966) were avoided. We could not find information in the literature on the Zn2+ or Cd2+ binding properties of the buffers used. Therefore, we measured the binding constants for a number of buffers, according to the method described by Good et al. 1966. This consisted of titrating the buffer alone, and then together with an equimolar amount of the metal salt (usually 10 mmol in a 100-ml vol). The binding constant was calculated from the relationship (): 1\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K{\mathrm{^{\prime}}}_{{\mathrm{M}}}=\frac{2 \left \left(\displaystyle\frac{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }{K_{{\mathrm{a}}}}-1\right) \right }{ \left \left[{\mathrm{B}}\right] \right \left \left(\displaystyle\frac{K_{{\mathrm{a}}}}{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }+1\right) \right }{\mathrm{,}}\end{equation*}\end{document} where K ′M is the metal binding constant, Ka is the proton dissociation constant defined in Fig. 3 (−pKa value), [H+M] is the H+ concentration at the midpoint of the titration curve in the presence of the metal being tested and [B] is the total buffer concentration. The higher the affinity of the buffer for metal, the greater the shift in the titration curve. Table gives the results.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus