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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 intracellular ZnCl2 on H+ currents in an inside-out patch studied at pH 6.5//6.5. The first family (A) was recorded within 5 min after forming the inside-out patch. The family in B was recorded starting 2.5 min after addition of 2.5 mM ZnCl2, and the family in C was recorded starting 1.5 min after washout. In all parts, the cell was held at −40 mV, and 16-s pulses were applied in 20-mV increments. Calibration bars in A apply to all families.
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Figure 10: Effects of intracellular ZnCl2 on H+ currents in an inside-out patch studied at pH 6.5//6.5. The first family (A) was recorded within 5 min after forming the inside-out patch. The family in B was recorded starting 2.5 min after addition of 2.5 mM ZnCl2, and the family in C was recorded starting 1.5 min after washout. In all parts, the cell was held at −40 mV, and 16-s pulses were applied in 20-mV increments. Calibration bars in A apply to all families.

Mentions: Inside-out patches were studied at pHo 7.5 or 6.5 (pipette pH) and pHi 6.5 (bath pH). Addition of 2.5 mM ZnCl2 to the bath (∼170 μM free Zn2+) reduced the H+ current amplitude (Fig. 10 B). This effect of ZnCl2 was reversible upon washout (Fig. 10 C). The reduction of H+ currents was similar to that observed in whole-cells dialyzed with ZnCl2 containing pipette solutions (Fig. 8 D), suggesting that similar concentrations were reached in the whole-cell experiments. There was no clear shift of the voltage dependence of gating. If anything, there was sometimes a small shift to more negative voltages. A small hyperpolarizing shift might be explainable by the slight lowering of pH after addition of ZnCl2 to the solution (0.023 U calculated, 0.05 U measured), due to displacement of protons from buffer. In some inside-out patches, the H+ currents decreased progressively and gradually after addition of ZnCl2. Spontaneous rundown may account for this largely irreversible loss of H+ current. In summary, the inside-out patch data support the conclusion that effects of internally applied ZnCl2 differ qualitatively as well as quantitatively from those of externally applied ZnCl2. Internal application of high concentrations of ZnCl2 produces only modest effects.


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 intracellular ZnCl2 on H+ currents in an inside-out patch studied at pH 6.5//6.5. The first family (A) was recorded within 5 min after forming the inside-out patch. The family in B was recorded starting 2.5 min after addition of 2.5 mM ZnCl2, and the family in C was recorded starting 1.5 min after washout. In all parts, the cell was held at −40 mV, and 16-s pulses were applied in 20-mV increments. Calibration bars in A apply to all families.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Effects of intracellular ZnCl2 on H+ currents in an inside-out patch studied at pH 6.5//6.5. The first family (A) was recorded within 5 min after forming the inside-out patch. The family in B was recorded starting 2.5 min after addition of 2.5 mM ZnCl2, and the family in C was recorded starting 1.5 min after washout. In all parts, the cell was held at −40 mV, and 16-s pulses were applied in 20-mV increments. Calibration bars in A apply to all families.
Mentions: Inside-out patches were studied at pHo 7.5 or 6.5 (pipette pH) and pHi 6.5 (bath pH). Addition of 2.5 mM ZnCl2 to the bath (∼170 μM free Zn2+) reduced the H+ current amplitude (Fig. 10 B). This effect of ZnCl2 was reversible upon washout (Fig. 10 C). The reduction of H+ currents was similar to that observed in whole-cells dialyzed with ZnCl2 containing pipette solutions (Fig. 8 D), suggesting that similar concentrations were reached in the whole-cell experiments. There was no clear shift of the voltage dependence of gating. If anything, there was sometimes a small shift to more negative voltages. A small hyperpolarizing shift might be explainable by the slight lowering of pH after addition of ZnCl2 to the solution (0.023 U calculated, 0.05 U measured), due to displacement of protons from buffer. In some inside-out patches, the H+ currents decreased progressively and gradually after addition of ZnCl2. Spontaneous rundown may account for this largely irreversible loss of H+ current. In summary, the inside-out patch data support the conclusion that effects of internally applied ZnCl2 differ qualitatively as well as quantitatively from those of externally applied ZnCl2. Internal application of high concentrations of ZnCl2 produces only modest effects.

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