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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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Optimized model predictions for the effect of ZnCl2 on the gH-V relationship (shown in Fig. 7).  is assumed for reasons described in the text, but with somewhat different parameters than in Fig. 11, where the τact effect is modeled: pKM is 8.0, pKa is 7.0, and a = 0.01. The model implies that the external Zn2+ receptor consists of three protonation sites, and that protonation of one site reduces the affinity of Zn2+ for the receptor 100-fold.
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Figure 12: Optimized model predictions for the effect of ZnCl2 on the gH-V relationship (shown in Fig. 7). is assumed for reasons described in the text, but with somewhat different parameters than in Fig. 11, where the τact effect is modeled: pKM is 8.0, pKa is 7.0, and a = 0.01. The model implies that the external Zn2+ receptor consists of three protonation sites, and that protonation of one site reduces the affinity of Zn2+ for the receptor 100-fold.

Mentions: The effects of ZnCl2 on the gH-V relationship were modeled in a similar manner as the effects on τact. Fig. 12 shows the predictions of Model 6 (as in Fig. 11), with parameters adjusted to match the ZnCl2 data from Fig. 7, which are superimposed. Several differences in the gH-V data compared with the τact data (Fig. 11) required different parameters. It was necessary to assume that more than two protonation sites were involved because the shift between pHo 6 and 5 was ∼230, whereas 100 is the maximum possible shift for a two-site model. pKM in all equations is defined by the metal concentration–response relationship at high pHo, where binding is unaffected by pH. pKa is somewhat model dependent, and is defined by the pHo at which the interaction between metal and H+ saturates. For a given model, this is set by the size of the shift in the high pHo region. Thus, in Fig. 12, pKa is 7.0 because this produces a sixfold shift between pHo 8 and 7, as observed in the data. Finally, it was necessary to assume some interaction between binding sites, because pure competition in a three-site model predicts too large a shift at low pHo. The value of the interaction factor, a, is established by the entire shift over the pHo range from 8 to 5. This shift was 105 in the data, and a = 0.01 matched this value. Setting a = 0.02 reduced the range to 3 × 104 and at a = 0 (pure competition) the range was too large, 4 × 105. The mechanistic interpretation is that protonation of one of the sites lowers the affinity of the Zn2+ receptor 100-fold. Assuming the same model, the affinity of Cd2+ for the external metal receptor is lower than that of Zn2+ by ∼2 U (roughly pKM 6).


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)

Optimized model predictions for the effect of ZnCl2 on the gH-V relationship (shown in Fig. 7).  is assumed for reasons described in the text, but with somewhat different parameters than in Fig. 11, where the τact effect is modeled: pKM is 8.0, pKa is 7.0, and a = 0.01. The model implies that the external Zn2+ receptor consists of three protonation sites, and that protonation of one site reduces the affinity of Zn2+ for the receptor 100-fold.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Optimized model predictions for the effect of ZnCl2 on the gH-V relationship (shown in Fig. 7). is assumed for reasons described in the text, but with somewhat different parameters than in Fig. 11, where the τact effect is modeled: pKM is 8.0, pKa is 7.0, and a = 0.01. The model implies that the external Zn2+ receptor consists of three protonation sites, and that protonation of one site reduces the affinity of Zn2+ for the receptor 100-fold.
Mentions: The effects of ZnCl2 on the gH-V relationship were modeled in a similar manner as the effects on τact. Fig. 12 shows the predictions of Model 6 (as in Fig. 11), with parameters adjusted to match the ZnCl2 data from Fig. 7, which are superimposed. Several differences in the gH-V data compared with the τact data (Fig. 11) required different parameters. It was necessary to assume that more than two protonation sites were involved because the shift between pHo 6 and 5 was ∼230, whereas 100 is the maximum possible shift for a two-site model. pKM in all equations is defined by the metal concentration–response relationship at high pHo, where binding is unaffected by pH. pKa is somewhat model dependent, and is defined by the pHo at which the interaction between metal and H+ saturates. For a given model, this is set by the size of the shift in the high pHo region. Thus, in Fig. 12, pKa is 7.0 because this produces a sixfold shift between pHo 8 and 7, as observed in the data. Finally, it was necessary to assume some interaction between binding sites, because pure competition in a three-site model predicts too large a shift at low pHo. The value of the interaction factor, a, is established by the entire shift over the pHo range from 8 to 5. This shift was 105 in the data, and a = 0.01 matched this value. Setting a = 0.02 reduced the range to 3 × 104 and at a = 0 (pure competition) the range was too large, 4 × 105. The mechanistic interpretation is that protonation of one of the sites lowers the affinity of the Zn2+ receptor 100-fold. Assuming the same model, the affinity of Cd2+ for the external metal receptor is lower than that of Zn2+ by ∼2 U (roughly pKM 6).

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