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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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High concentrations of intracellular ZnCl2 have only subtle effects on H+ currents. The families of H+ currents at pH 6.5//6.5 were recorded in a control cell (A) and with 2.5 mM ZnCl2 in the pipette solution (B), and are scaled according to membrane capacity. The measured Vrev was +4 mV in A and +1 mV in B. Note the slower tail current decay with ZnCl2 in the pipette. Considering binding of ZnCl2 to 1 mM EGTA and 100 mM BisTris in the pipette solution, the free [Zn2+] was ∼170 μM. (C) Identical 8-s pulses to +50 mV were applied before and after addition of the pipette solution used in B to the bath, in the same cell shown in A, demonstrating the dramatic effects of this solution applied externally. The threshold for activating outward H+ current shifted from +30 to +60 mV after adding ZnCl2 to the bath (determined using high gain and 5-mV increments). (D) Average (mean ± SEM) H+ current–voltage relationships normalized according to membrane capacity, in 6–10 control cells (•), 9–14 cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪), and 3 cells studied with 2.5 mM CdCl2 in the pipette solution (⋄), all at pH 6.5//6.5. *Values for ZnCl2 at +80 and +100 mV differ significantly from control (P < 0.05).
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Figure 8: High concentrations of intracellular ZnCl2 have only subtle effects on H+ currents. The families of H+ currents at pH 6.5//6.5 were recorded in a control cell (A) and with 2.5 mM ZnCl2 in the pipette solution (B), and are scaled according to membrane capacity. The measured Vrev was +4 mV in A and +1 mV in B. Note the slower tail current decay with ZnCl2 in the pipette. Considering binding of ZnCl2 to 1 mM EGTA and 100 mM BisTris in the pipette solution, the free [Zn2+] was ∼170 μM. (C) Identical 8-s pulses to +50 mV were applied before and after addition of the pipette solution used in B to the bath, in the same cell shown in A, demonstrating the dramatic effects of this solution applied externally. The threshold for activating outward H+ current shifted from +30 to +60 mV after adding ZnCl2 to the bath (determined using high gain and 5-mV increments). (D) Average (mean ± SEM) H+ current–voltage relationships normalized according to membrane capacity, in 6–10 control cells (•), 9–14 cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪), and 3 cells studied with 2.5 mM CdCl2 in the pipette solution (⋄), all at pH 6.5//6.5. *Values for ZnCl2 at +80 and +100 mV differ significantly from control (P < 0.05).

Mentions: Effects of internally applied ZnCl2 were studied in the whole-cell configuration and in inside-out patches. Fig. 8 illustrates families of H+ currents in cells studied at pH 6.5//6.5 without (A) and with (B) 2.5 mM ZnCl2 added to the pipette solution. The H+ currents appear generally similar, although closer inspection reveals that the tail currents decayed more slowly in the cell with internal ZnCl2. The pipette solution contained 1 mM EGTA and BisTris buffer (which will bind ∼90% of the Zn2+ under these conditions, Table ), so the addition of 2.5 mM ZnCl2 results in a free [Zn2+] ∼170 μM. Fig. 8 C illustrates that addition of the pH 6.5 ZnCl2 containing pipette solution to the bath dramatically reduced the H+ current at +50 mV. This result makes it clear that ZnCl2 applied externally is much more effective than when applied internally. Several cells were studied with 2.5 mM ZnCl2 in the pipette at pHi 7.5. HEPES buffer does not bind ZnCl2 detectably (Table ), hence the free [ZnCl2] was ∼1.5 mM. In these cells, the H+ currents also appeared normal (data not shown).


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)

High concentrations of intracellular ZnCl2 have only subtle effects on H+ currents. The families of H+ currents at pH 6.5//6.5 were recorded in a control cell (A) and with 2.5 mM ZnCl2 in the pipette solution (B), and are scaled according to membrane capacity. The measured Vrev was +4 mV in A and +1 mV in B. Note the slower tail current decay with ZnCl2 in the pipette. Considering binding of ZnCl2 to 1 mM EGTA and 100 mM BisTris in the pipette solution, the free [Zn2+] was ∼170 μM. (C) Identical 8-s pulses to +50 mV were applied before and after addition of the pipette solution used in B to the bath, in the same cell shown in A, demonstrating the dramatic effects of this solution applied externally. The threshold for activating outward H+ current shifted from +30 to +60 mV after adding ZnCl2 to the bath (determined using high gain and 5-mV increments). (D) Average (mean ± SEM) H+ current–voltage relationships normalized according to membrane capacity, in 6–10 control cells (•), 9–14 cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪), and 3 cells studied with 2.5 mM CdCl2 in the pipette solution (⋄), all at pH 6.5//6.5. *Values for ZnCl2 at +80 and +100 mV differ significantly from control (P < 0.05).
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Figure 8: High concentrations of intracellular ZnCl2 have only subtle effects on H+ currents. The families of H+ currents at pH 6.5//6.5 were recorded in a control cell (A) and with 2.5 mM ZnCl2 in the pipette solution (B), and are scaled according to membrane capacity. The measured Vrev was +4 mV in A and +1 mV in B. Note the slower tail current decay with ZnCl2 in the pipette. Considering binding of ZnCl2 to 1 mM EGTA and 100 mM BisTris in the pipette solution, the free [Zn2+] was ∼170 μM. (C) Identical 8-s pulses to +50 mV were applied before and after addition of the pipette solution used in B to the bath, in the same cell shown in A, demonstrating the dramatic effects of this solution applied externally. The threshold for activating outward H+ current shifted from +30 to +60 mV after adding ZnCl2 to the bath (determined using high gain and 5-mV increments). (D) Average (mean ± SEM) H+ current–voltage relationships normalized according to membrane capacity, in 6–10 control cells (•), 9–14 cells studied with 2.5 mM ZnCl2 added to the pipette solution (▪), and 3 cells studied with 2.5 mM CdCl2 in the pipette solution (⋄), all at pH 6.5//6.5. *Values for ZnCl2 at +80 and +100 mV differ significantly from control (P < 0.05).
Mentions: Effects of internally applied ZnCl2 were studied in the whole-cell configuration and in inside-out patches. Fig. 8 illustrates families of H+ currents in cells studied at pH 6.5//6.5 without (A) and with (B) 2.5 mM ZnCl2 added to the pipette solution. The H+ currents appear generally similar, although closer inspection reveals that the tail currents decayed more slowly in the cell with internal ZnCl2. The pipette solution contained 1 mM EGTA and BisTris buffer (which will bind ∼90% of the Zn2+ under these conditions, Table ), so the addition of 2.5 mM ZnCl2 results in a free [Zn2+] ∼170 μM. Fig. 8 C illustrates that addition of the pH 6.5 ZnCl2 containing pipette solution to the bath dramatically reduced the H+ current at +50 mV. This result makes it clear that ZnCl2 applied externally is much more effective than when applied internally. Several cells were studied with 2.5 mM ZnCl2 in the pipette at pHi 7.5. HEPES buffer does not bind ZnCl2 detectably (Table ), hence the free [ZnCl2] was ∼1.5 mM. In these cells, the H+ currents also appeared normal (data not shown).

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