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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 divalent metals on the gH-V relationship are incompatible with the idea of voltage-dependent block. (A) The gH-V relationships in a cell studied in the presence of several metals: controls (dashed lines), 0.1 mM CdCl2 (•), 1 mM CdCl2 (▾), 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄). The sequence was control, all CdCl2 concentrations, control, NiCl2, ZnCl2, and control. (B) The same gH-V relationships in A shifted along the voltage axis so that they superimpose at small gH. Other than small differences in the limiting gH,max, the shape of the voltage dependence appears similar. The voltage shifts applied were: 0, +8, and +34 mV for 0.1, 1.0, and 10 mM CdCl2, respectively, +15 mV for NiCl2 and +18 mV for ZnCl2. (C) The same gH-V relationships plotted on linear axes and scaled to have similar gH,max appear to simply shift along the voltage axis. The scale factor was determined by taking the ratio of gH in the presence of metal to that at +80 mV in the first control measurement. To compensate for the apparent voltage shift (compare A and B), the gH value used for this purpose for the metal data was shifted by 10 mV (1 mM CdCl2, 10 mM NiCl2), 20 mV (ZnCl2), or 30 mV (10 mM CdCl2). All scale factors were <2.5. (D) The steepness of the apparent voltage dependence of “block” by divalent cations is similar to that of the gH-V relationship itself. The data in C are plotted as a ratio of the gH in the presence of metal to that in its absence, at each voltage, using the same symbols as other parts of this figure. There is no block at any voltage at 0.1 mM CdCl2 (•). The control gH-V relationship (C, dashed line) was fitted to a simple Boltzmann distribution and normalized to its fitted maximum. The slope factors of Boltzmann fits were 12.5 mV for control, and for metal ranged from 8 to 13 mV in fits constrained to limit at 1.0.
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Figure 3: Effects of divalent metals on the gH-V relationship are incompatible with the idea of voltage-dependent block. (A) The gH-V relationships in a cell studied in the presence of several metals: controls (dashed lines), 0.1 mM CdCl2 (•), 1 mM CdCl2 (▾), 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄). The sequence was control, all CdCl2 concentrations, control, NiCl2, ZnCl2, and control. (B) The same gH-V relationships in A shifted along the voltage axis so that they superimpose at small gH. Other than small differences in the limiting gH,max, the shape of the voltage dependence appears similar. The voltage shifts applied were: 0, +8, and +34 mV for 0.1, 1.0, and 10 mM CdCl2, respectively, +15 mV for NiCl2 and +18 mV for ZnCl2. (C) The same gH-V relationships plotted on linear axes and scaled to have similar gH,max appear to simply shift along the voltage axis. The scale factor was determined by taking the ratio of gH in the presence of metal to that at +80 mV in the first control measurement. To compensate for the apparent voltage shift (compare A and B), the gH value used for this purpose for the metal data was shifted by 10 mV (1 mM CdCl2, 10 mM NiCl2), 20 mV (ZnCl2), or 30 mV (10 mM CdCl2). All scale factors were <2.5. (D) The steepness of the apparent voltage dependence of “block” by divalent cations is similar to that of the gH-V relationship itself. The data in C are plotted as a ratio of the gH in the presence of metal to that in its absence, at each voltage, using the same symbols as other parts of this figure. There is no block at any voltage at 0.1 mM CdCl2 (•). The control gH-V relationship (C, dashed line) was fitted to a simple Boltzmann distribution and normalized to its fitted maximum. The slope factors of Boltzmann fits were 12.5 mV for control, and for metal ranged from 8 to 13 mV in fits constrained to limit at 1.0.

Mentions: The effects of ZnCl2 and other metals might reflect voltage-independent interaction of the metal with the channel or nearby membrane. By binding to or screening negative charges near the external side of the H+ channel, metals could bias the membrane potential sensed by the channel's voltage sensor (Frankenhaeuser and Hodgkin 1957). In the simplest scenario, the voltage-dependent properties of the channel will simply shift along the voltage axis. Fig. 3 A illustrates proton chord conductance (gH)–V relationships in one cell in the absence (dashed lines) or presence of 100 μM ZnCl2 (⋄), 10 mM NiCl2 (▵), or several concentrations of CdCl2 (solid symbols). When shifted along the voltage axis, the gH-V relationships appear quite similar (Fig. 3 B), consistent with this mechanism. These metals may reduce the limiting gH (gH,max) slightly, although for the data shown here this effect was smaller than the variability in the control measurements. At higher metal concentrations, some reduction in gH,max usually became evident, but was difficult to measure accurately. In Fig. 3 C, the gH-V relationships are plotted on linear axes, scaled to the same gH,max to illustrate their similar shape and slope. The predominant effect is a simple voltage shift.


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 divalent metals on the gH-V relationship are incompatible with the idea of voltage-dependent block. (A) The gH-V relationships in a cell studied in the presence of several metals: controls (dashed lines), 0.1 mM CdCl2 (•), 1 mM CdCl2 (▾), 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄). The sequence was control, all CdCl2 concentrations, control, NiCl2, ZnCl2, and control. (B) The same gH-V relationships in A shifted along the voltage axis so that they superimpose at small gH. Other than small differences in the limiting gH,max, the shape of the voltage dependence appears similar. The voltage shifts applied were: 0, +8, and +34 mV for 0.1, 1.0, and 10 mM CdCl2, respectively, +15 mV for NiCl2 and +18 mV for ZnCl2. (C) The same gH-V relationships plotted on linear axes and scaled to have similar gH,max appear to simply shift along the voltage axis. The scale factor was determined by taking the ratio of gH in the presence of metal to that at +80 mV in the first control measurement. To compensate for the apparent voltage shift (compare A and B), the gH value used for this purpose for the metal data was shifted by 10 mV (1 mM CdCl2, 10 mM NiCl2), 20 mV (ZnCl2), or 30 mV (10 mM CdCl2). All scale factors were <2.5. (D) The steepness of the apparent voltage dependence of “block” by divalent cations is similar to that of the gH-V relationship itself. The data in C are plotted as a ratio of the gH in the presence of metal to that in its absence, at each voltage, using the same symbols as other parts of this figure. There is no block at any voltage at 0.1 mM CdCl2 (•). The control gH-V relationship (C, dashed line) was fitted to a simple Boltzmann distribution and normalized to its fitted maximum. The slope factors of Boltzmann fits were 12.5 mV for control, and for metal ranged from 8 to 13 mV in fits constrained to limit at 1.0.
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Related In: Results  -  Collection

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Figure 3: Effects of divalent metals on the gH-V relationship are incompatible with the idea of voltage-dependent block. (A) The gH-V relationships in a cell studied in the presence of several metals: controls (dashed lines), 0.1 mM CdCl2 (•), 1 mM CdCl2 (▾), 10 mM CdCl2 (▪), 10 mM NiCl2 (▵), and 0.1 mM ZnCl2 (⋄). The sequence was control, all CdCl2 concentrations, control, NiCl2, ZnCl2, and control. (B) The same gH-V relationships in A shifted along the voltage axis so that they superimpose at small gH. Other than small differences in the limiting gH,max, the shape of the voltage dependence appears similar. The voltage shifts applied were: 0, +8, and +34 mV for 0.1, 1.0, and 10 mM CdCl2, respectively, +15 mV for NiCl2 and +18 mV for ZnCl2. (C) The same gH-V relationships plotted on linear axes and scaled to have similar gH,max appear to simply shift along the voltage axis. The scale factor was determined by taking the ratio of gH in the presence of metal to that at +80 mV in the first control measurement. To compensate for the apparent voltage shift (compare A and B), the gH value used for this purpose for the metal data was shifted by 10 mV (1 mM CdCl2, 10 mM NiCl2), 20 mV (ZnCl2), or 30 mV (10 mM CdCl2). All scale factors were <2.5. (D) The steepness of the apparent voltage dependence of “block” by divalent cations is similar to that of the gH-V relationship itself. The data in C are plotted as a ratio of the gH in the presence of metal to that in its absence, at each voltage, using the same symbols as other parts of this figure. There is no block at any voltage at 0.1 mM CdCl2 (•). The control gH-V relationship (C, dashed line) was fitted to a simple Boltzmann distribution and normalized to its fitted maximum. The slope factors of Boltzmann fits were 12.5 mV for control, and for metal ranged from 8 to 13 mV in fits constrained to limit at 1.0.
Mentions: The effects of ZnCl2 and other metals might reflect voltage-independent interaction of the metal with the channel or nearby membrane. By binding to or screening negative charges near the external side of the H+ channel, metals could bias the membrane potential sensed by the channel's voltage sensor (Frankenhaeuser and Hodgkin 1957). In the simplest scenario, the voltage-dependent properties of the channel will simply shift along the voltage axis. Fig. 3 A illustrates proton chord conductance (gH)–V relationships in one cell in the absence (dashed lines) or presence of 100 μM ZnCl2 (⋄), 10 mM NiCl2 (▵), or several concentrations of CdCl2 (solid symbols). When shifted along the voltage axis, the gH-V relationships appear quite similar (Fig. 3 B), consistent with this mechanism. These metals may reduce the limiting gH (gH,max) slightly, although for the data shown here this effect was smaller than the variability in the control measurements. At higher metal concentrations, some reduction in gH,max usually became evident, but was difficult to measure accurately. In Fig. 3 C, the gH-V relationships are plotted on linear axes, scaled to the same gH,max to illustrate their similar shape and slope. The predominant effect is a simple voltage shift.

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