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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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Effects of metals on the activation time constant, τact, in the same cell studied at pH 6.0//5.5 as in Fig. 3. (A) The voltage dependence of τact is plotted in the presence of CdCl2, NiCl2, and ZnCl2 at concentrations indicated in the figure (mM). Four control data sets are plotted as dashed lines. The sequence was control twice, CdCl2, control, NiCl2, ZnCl2, and control. (B) The data in the presence of metals is shifted to more negative voltages, according to the shift of the gH-V relationship observed in this cell (in Fig. 3). For clarity, only data at 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄) are plotted. Note that the slowing of τact by CdCl2 and NiCl2 appears ascribable to a simple voltage shift, whereas ZnCl2 has an additional slowing effect.
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Figure 4: Effects of metals on the activation time constant, τact, in the same cell studied at pH 6.0//5.5 as in Fig. 3. (A) The voltage dependence of τact is plotted in the presence of CdCl2, NiCl2, and ZnCl2 at concentrations indicated in the figure (mM). Four control data sets are plotted as dashed lines. The sequence was control twice, CdCl2, control, NiCl2, ZnCl2, and control. (B) The data in the presence of metals is shifted to more negative voltages, according to the shift of the gH-V relationship observed in this cell (in Fig. 3). For clarity, only data at 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄) are plotted. Note that the slowing of τact by CdCl2 and NiCl2 appears ascribable to a simple voltage shift, whereas ZnCl2 has an additional slowing effect.

Mentions: A prominent effect of ZnCl2 is to slow the activation of H+ currents. We quantified this effect by fitting the turn-on of current during depolarizing pulses to a single exponential, after a delay. This procedure provides a reasonable fit under most conditions. In the presence of ZnCl2, both the delay and τact were increased by roughly the same factor. We focussed mainly on metal effects on τact, which are illustrated in Fig. 4 for the same cell shown in Fig. 3. Because the τact-V relationship is nearly exponential (linear on semi-log axes), it is not possible to distinguish whether τact is slowed or its voltage dependence is shifted, or both. In the simplest case of a Huxley-Frankenhaeuser-Hodgkin voltage shift, all kinetic parameters should be shifted equally along the voltage axis. To explore the extent to which this model might apply, the τact data in Fig. 4 B were “corrected” by the voltage shift determined for the gH-V relationship (Fig. 3 B). To a rough approximation, the τact effect in CdCl2 and NiCl2 appears to be explainable by this simple voltage shift. Closer examination of Fig. 4 B and other data (not shown) at high CdCl2 concentrations indicates that CdCl2 slows activation somewhat more than is accounted for by the shift of the gH-V relationship, consistent with a previous study of CdCl2 on H+ currents (Byerly et al. 1984). In contrast, ZnCl2 slows channel opening dramatically, and far beyond its shift of the gH-V relationship. The effects of ZnCl2 are dominated by an interaction with the H+ channel that results in τact slowing, beyond a simple voltage shift of all parameters.


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)

Effects of metals on the activation time constant, τact, in the same cell studied at pH 6.0//5.5 as in Fig. 3. (A) The voltage dependence of τact is plotted in the presence of CdCl2, NiCl2, and ZnCl2 at concentrations indicated in the figure (mM). Four control data sets are plotted as dashed lines. The sequence was control twice, CdCl2, control, NiCl2, ZnCl2, and control. (B) The data in the presence of metals is shifted to more negative voltages, according to the shift of the gH-V relationship observed in this cell (in Fig. 3). For clarity, only data at 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄) are plotted. Note that the slowing of τact by CdCl2 and NiCl2 appears ascribable to a simple voltage shift, whereas ZnCl2 has an additional slowing effect.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Effects of metals on the activation time constant, τact, in the same cell studied at pH 6.0//5.5 as in Fig. 3. (A) The voltage dependence of τact is plotted in the presence of CdCl2, NiCl2, and ZnCl2 at concentrations indicated in the figure (mM). Four control data sets are plotted as dashed lines. The sequence was control twice, CdCl2, control, NiCl2, ZnCl2, and control. (B) The data in the presence of metals is shifted to more negative voltages, according to the shift of the gH-V relationship observed in this cell (in Fig. 3). For clarity, only data at 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄) are plotted. Note that the slowing of τact by CdCl2 and NiCl2 appears ascribable to a simple voltage shift, whereas ZnCl2 has an additional slowing effect.
Mentions: A prominent effect of ZnCl2 is to slow the activation of H+ currents. We quantified this effect by fitting the turn-on of current during depolarizing pulses to a single exponential, after a delay. This procedure provides a reasonable fit under most conditions. In the presence of ZnCl2, both the delay and τact were increased by roughly the same factor. We focussed mainly on metal effects on τact, which are illustrated in Fig. 4 for the same cell shown in Fig. 3. Because the τact-V relationship is nearly exponential (linear on semi-log axes), it is not possible to distinguish whether τact is slowed or its voltage dependence is shifted, or both. In the simplest case of a Huxley-Frankenhaeuser-Hodgkin voltage shift, all kinetic parameters should be shifted equally along the voltage axis. To explore the extent to which this model might apply, the τact data in Fig. 4 B were “corrected” by the voltage shift determined for the gH-V relationship (Fig. 3 B). To a rough approximation, the τact effect in CdCl2 and NiCl2 appears to be explainable by this simple voltage shift. Closer examination of Fig. 4 B and other data (not shown) at high CdCl2 concentrations indicates that CdCl2 slows activation somewhat more than is accounted for by the shift of the gH-V relationship, consistent with a previous study of CdCl2 on H+ currents (Byerly et al. 1984). In contrast, ZnCl2 slows channel opening dramatically, and far beyond its shift of the gH-V relationship. The effects of ZnCl2 are dominated by an interaction with the H+ channel that results in τact slowing, beyond a simple voltage shift of all parameters.

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