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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 the mean (±SEM) activation time constant, τact, and deactivation time constant, τtail, in cells studied at pH 6.5//6.5. Tail current decay was fitted with a single exponential in three to nine control cells (○) and in four to eight cells studied with 2.5 mM ZnCl2 added to the pipette solution (•). All τtail values with ZnCl2 in the pipette differ significantly from control (P < 0.01). Values of τact obtained by fitting a single exponential after a delay are plotted from 10–12 control cells (□) and from four to nine cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪).
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Figure 9: Effects of intracellular ZnCl2 on the mean (±SEM) activation time constant, τact, and deactivation time constant, τtail, in cells studied at pH 6.5//6.5. Tail current decay was fitted with a single exponential in three to nine control cells (○) and in four to eight cells studied with 2.5 mM ZnCl2 added to the pipette solution (•). All τtail values with ZnCl2 in the pipette differ significantly from control (P < 0.01). Values of τact obtained by fitting a single exponential after a delay are plotted from 10–12 control cells (□) and from four to nine cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪).

Mentions: Fig. 9 illustrates mean τact values in cells studied at pH 6.5//6.5 with (▪) and without (□) 2.5 mM ZnCl2 in the pipette solution. No difference in the kinetics of H+ current activation was detected. However, channel closing was significantly slower in cells studied with internal ZnCl2. Fig. 9 shows mean values of τtail in cells studied with internal ZnCl2 (•) and in control cells (○). The deactivation rate on average was 3.1-fold slower with internal ZnCl2 (measured between −50 and +10 mV). In three cells studied with 2.5 mM CdCl2 added to the pipette solutions, the average slowing of τtail was 1.8-fold at 10 voltages from −80 to +20 mV (P < 0.05 at each voltage) (not shown). Applied internally, ZnCl2 thus slows closing without affecting activation. In contrast, externally applied ZnCl2 slowed activation and, if anything, accelerated deactivation. Clearly the internal and external sites of action of ZnCl2 are functionally quite different.


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 the mean (±SEM) activation time constant, τact, and deactivation time constant, τtail, in cells studied at pH 6.5//6.5. Tail current decay was fitted with a single exponential in three to nine control cells (○) and in four to eight cells studied with 2.5 mM ZnCl2 added to the pipette solution (•). All τtail values with ZnCl2 in the pipette differ significantly from control (P < 0.01). Values of τact obtained by fitting a single exponential after a delay are plotted from 10–12 control cells (□) and from four to nine cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Effects of intracellular ZnCl2 on the mean (±SEM) activation time constant, τact, and deactivation time constant, τtail, in cells studied at pH 6.5//6.5. Tail current decay was fitted with a single exponential in three to nine control cells (○) and in four to eight cells studied with 2.5 mM ZnCl2 added to the pipette solution (•). All τtail values with ZnCl2 in the pipette differ significantly from control (P < 0.01). Values of τact obtained by fitting a single exponential after a delay are plotted from 10–12 control cells (□) and from four to nine cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪).
Mentions: Fig. 9 illustrates mean τact values in cells studied at pH 6.5//6.5 with (▪) and without (□) 2.5 mM ZnCl2 in the pipette solution. No difference in the kinetics of H+ current activation was detected. However, channel closing was significantly slower in cells studied with internal ZnCl2. Fig. 9 shows mean values of τtail in cells studied with internal ZnCl2 (•) and in control cells (○). The deactivation rate on average was 3.1-fold slower with internal ZnCl2 (measured between −50 and +10 mV). In three cells studied with 2.5 mM CdCl2 added to the pipette solutions, the average slowing of τtail was 1.8-fold at 10 voltages from −80 to +20 mV (P < 0.05 at each voltage) (not shown). Applied internally, ZnCl2 thus slows closing without affecting activation. In contrast, externally applied ZnCl2 slowed activation and, if anything, accelerated deactivation. Clearly the internal and external sites of action of ZnCl2 are functionally quite different.

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