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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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The probability that the metal receptor is occupied by a metal ion as predicted by various models with different assumptions about the interaction between H+ and Zn2+. To relate the occupancy of the metal receptor to data such as in Fig. 6, it is necessary to assume a specific mechanism, as discussed in the text. On the other hand, the pKa value is set by the pHo at which the effect saturates, and is relatively insensitive to this relationship. Binding constants for H+ and Zn2+, pKa, and pKM, respectively, are defined in the text. (A) Model 1 (pKa 6.6 and pKM 6.5, from ) assumes simple competition between H+ and Zn2+ for a single site. At low pH, the concentration of Zn2+ required to produce a given occupancy increases 10-fold per unit decrease in pH. (B) Model 2 (pKa 6.6 and pKM 6.5, from ) describes noncompetitive inhibition, in which H+ and Zn2+ bind independently to separate sites. These curves superimpose if scaled up. (C) Model 3 (pKa 6.6 and pKM 6.5, from ) assumes that the effect is the same whether H+ or Zn2+ is bound to the site. If the pH 5 curve is scaled to the pH 9 curve, the former is shifted to the right ∼10-fold. (D) Model 4 (pKa 6.6 and pKM 6.5, from ) assumes two identical protonation sites that combine to coordinate one Zn2+ ion. Pure competition is assumed, in that protonation of either site prevents Zn2+ binding. (E) Model 5 (pKa 6.2 and pKM 6.5, from ) is identical to Model 4 except that there are three independent sites, protonation of any of which prevents Zn2+ binding. The pKa was adjusted to reproduce the 10-fold change in apparent potency of Zn2+ observed between pH 6 and 7 (Fig. 6), but the apparent potency of Zn2+ changes too much between pH 6 and 5 (∼285-fold, compared with 100-fold for the data in Fig. 6), and ∼850-fold between pH 5 and 4, approaching the low pH limit of a 1,000-fold change. (F) Model 6 (pKa 6.3, pKM 6.5, and a = 0.03, from ) assumes three sites, but the protonation of one site lowers the affinity of the next site(s) to 0.03 of the original affinity. In essence, the fully protonated channel has a much lower affinity for Zn2+ (apparent pKM ∼ 1.3) so that the effect of pH on Zn2+ saturates at either high or low pH. With this interaction factor (a = 0.03), decreasing pH from 4 to 3 lowers the Zn2+ affinity less than twofold, and lowering pH to <3 has no further effect.
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Figure 13: The probability that the metal receptor is occupied by a metal ion as predicted by various models with different assumptions about the interaction between H+ and Zn2+. To relate the occupancy of the metal receptor to data such as in Fig. 6, it is necessary to assume a specific mechanism, as discussed in the text. On the other hand, the pKa value is set by the pHo at which the effect saturates, and is relatively insensitive to this relationship. Binding constants for H+ and Zn2+, pKa, and pKM, respectively, are defined in the text. (A) Model 1 (pKa 6.6 and pKM 6.5, from ) assumes simple competition between H+ and Zn2+ for a single site. At low pH, the concentration of Zn2+ required to produce a given occupancy increases 10-fold per unit decrease in pH. (B) Model 2 (pKa 6.6 and pKM 6.5, from ) describes noncompetitive inhibition, in which H+ and Zn2+ bind independently to separate sites. These curves superimpose if scaled up. (C) Model 3 (pKa 6.6 and pKM 6.5, from ) assumes that the effect is the same whether H+ or Zn2+ is bound to the site. If the pH 5 curve is scaled to the pH 9 curve, the former is shifted to the right ∼10-fold. (D) Model 4 (pKa 6.6 and pKM 6.5, from ) assumes two identical protonation sites that combine to coordinate one Zn2+ ion. Pure competition is assumed, in that protonation of either site prevents Zn2+ binding. (E) Model 5 (pKa 6.2 and pKM 6.5, from ) is identical to Model 4 except that there are three independent sites, protonation of any of which prevents Zn2+ binding. The pKa was adjusted to reproduce the 10-fold change in apparent potency of Zn2+ observed between pH 6 and 7 (Fig. 6), but the apparent potency of Zn2+ changes too much between pH 6 and 5 (∼285-fold, compared with 100-fold for the data in Fig. 6), and ∼850-fold between pH 5 and 4, approaching the low pH limit of a 1,000-fold change. (F) Model 6 (pKa 6.3, pKM 6.5, and a = 0.03, from ) assumes three sites, but the protonation of one site lowers the affinity of the next site(s) to 0.03 of the original affinity. In essence, the fully protonated channel has a much lower affinity for Zn2+ (apparent pKM ∼ 1.3) so that the effect of pH on Zn2+ saturates at either high or low pH. With this interaction factor (a = 0.03), decreasing pH from 4 to 3 lowers the Zn2+ affinity less than twofold, and lowering pH to <3 has no further effect.

Mentions: The explores the predictions of several possible mechanisms of competition between Zn2+ and H+ for hypothetical binding sites on H+ channels. The pHo dependence of Zn2+ effects on τact are reasonably compatible with Models 4, 5, or 6 (see Fig. 13 for all models). These models assume that the external Zn2+ receptor on proton channels is formed by multiple protonation sites that are accessible to the external solution and that coordinate the binding of a single Zn2+. If H+ and Zn2+ compete directly for the same site(s), then at least two to three protonation sites must exist. If H+ and Zn2+ bind to different sites, then there must be substantial interaction between them, and the range of the pH dependence indicates that protonation of one site lowers the affinity of the remaining site(s) for Zn2+ by a factor ∼30. Similar binding constants reproduce the pH dependence of Zn2+ effects using any of several models: pKM is 6.5 and pKa is 6.2–6.6 and is somewhat model dependent.


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)

The probability that the metal receptor is occupied by a metal ion as predicted by various models with different assumptions about the interaction between H+ and Zn2+. To relate the occupancy of the metal receptor to data such as in Fig. 6, it is necessary to assume a specific mechanism, as discussed in the text. On the other hand, the pKa value is set by the pHo at which the effect saturates, and is relatively insensitive to this relationship. Binding constants for H+ and Zn2+, pKa, and pKM, respectively, are defined in the text. (A) Model 1 (pKa 6.6 and pKM 6.5, from ) assumes simple competition between H+ and Zn2+ for a single site. At low pH, the concentration of Zn2+ required to produce a given occupancy increases 10-fold per unit decrease in pH. (B) Model 2 (pKa 6.6 and pKM 6.5, from ) describes noncompetitive inhibition, in which H+ and Zn2+ bind independently to separate sites. These curves superimpose if scaled up. (C) Model 3 (pKa 6.6 and pKM 6.5, from ) assumes that the effect is the same whether H+ or Zn2+ is bound to the site. If the pH 5 curve is scaled to the pH 9 curve, the former is shifted to the right ∼10-fold. (D) Model 4 (pKa 6.6 and pKM 6.5, from ) assumes two identical protonation sites that combine to coordinate one Zn2+ ion. Pure competition is assumed, in that protonation of either site prevents Zn2+ binding. (E) Model 5 (pKa 6.2 and pKM 6.5, from ) is identical to Model 4 except that there are three independent sites, protonation of any of which prevents Zn2+ binding. The pKa was adjusted to reproduce the 10-fold change in apparent potency of Zn2+ observed between pH 6 and 7 (Fig. 6), but the apparent potency of Zn2+ changes too much between pH 6 and 5 (∼285-fold, compared with 100-fold for the data in Fig. 6), and ∼850-fold between pH 5 and 4, approaching the low pH limit of a 1,000-fold change. (F) Model 6 (pKa 6.3, pKM 6.5, and a = 0.03, from ) assumes three sites, but the protonation of one site lowers the affinity of the next site(s) to 0.03 of the original affinity. In essence, the fully protonated channel has a much lower affinity for Zn2+ (apparent pKM ∼ 1.3) so that the effect of pH on Zn2+ saturates at either high or low pH. With this interaction factor (a = 0.03), decreasing pH from 4 to 3 lowers the Zn2+ affinity less than twofold, and lowering pH to <3 has no further effect.
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Figure 13: The probability that the metal receptor is occupied by a metal ion as predicted by various models with different assumptions about the interaction between H+ and Zn2+. To relate the occupancy of the metal receptor to data such as in Fig. 6, it is necessary to assume a specific mechanism, as discussed in the text. On the other hand, the pKa value is set by the pHo at which the effect saturates, and is relatively insensitive to this relationship. Binding constants for H+ and Zn2+, pKa, and pKM, respectively, are defined in the text. (A) Model 1 (pKa 6.6 and pKM 6.5, from ) assumes simple competition between H+ and Zn2+ for a single site. At low pH, the concentration of Zn2+ required to produce a given occupancy increases 10-fold per unit decrease in pH. (B) Model 2 (pKa 6.6 and pKM 6.5, from ) describes noncompetitive inhibition, in which H+ and Zn2+ bind independently to separate sites. These curves superimpose if scaled up. (C) Model 3 (pKa 6.6 and pKM 6.5, from ) assumes that the effect is the same whether H+ or Zn2+ is bound to the site. If the pH 5 curve is scaled to the pH 9 curve, the former is shifted to the right ∼10-fold. (D) Model 4 (pKa 6.6 and pKM 6.5, from ) assumes two identical protonation sites that combine to coordinate one Zn2+ ion. Pure competition is assumed, in that protonation of either site prevents Zn2+ binding. (E) Model 5 (pKa 6.2 and pKM 6.5, from ) is identical to Model 4 except that there are three independent sites, protonation of any of which prevents Zn2+ binding. The pKa was adjusted to reproduce the 10-fold change in apparent potency of Zn2+ observed between pH 6 and 7 (Fig. 6), but the apparent potency of Zn2+ changes too much between pH 6 and 5 (∼285-fold, compared with 100-fold for the data in Fig. 6), and ∼850-fold between pH 5 and 4, approaching the low pH limit of a 1,000-fold change. (F) Model 6 (pKa 6.3, pKM 6.5, and a = 0.03, from ) assumes three sites, but the protonation of one site lowers the affinity of the next site(s) to 0.03 of the original affinity. In essence, the fully protonated channel has a much lower affinity for Zn2+ (apparent pKM ∼ 1.3) so that the effect of pH on Zn2+ saturates at either high or low pH. With this interaction factor (a = 0.03), decreasing pH from 4 to 3 lowers the Zn2+ affinity less than twofold, and lowering pH to <3 has no further effect.
Mentions: The explores the predictions of several possible mechanisms of competition between Zn2+ and H+ for hypothetical binding sites on H+ channels. The pHo dependence of Zn2+ effects on τact are reasonably compatible with Models 4, 5, or 6 (see Fig. 13 for all models). These models assume that the external Zn2+ receptor on proton channels is formed by multiple protonation sites that are accessible to the external solution and that coordinate the binding of a single Zn2+. If H+ and Zn2+ compete directly for the same site(s), then at least two to three protonation sites must exist. If H+ and Zn2+ bind to different sites, then there must be substantial interaction between them, and the range of the pH dependence indicates that protonation of one site lowers the affinity of the remaining site(s) for Zn2+ by a factor ∼30. Similar binding constants reproduce the pH dependence of Zn2+ effects using any of several models: pKM is 6.5 and pKa is 6.2–6.6 and is somewhat model dependent.

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