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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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(A) Instantaneous current–voltage relationships in a cell studied at pH 7.0//6.5 before (•) and after (▪) addition of 10 μM ZnCl2 to the bath. A prepulse to +40 mV for control and +100 mV in the presence of ZnCl2 was applied to open H+ channels, followed by a test pulse to the voltage on the abscissae. The current at the start of the test pulse, after the capacitive transient, is plotted. (B) Data from A after correction for the current at the end of the prepulse, and normalized to be equal at +100 mV. Dividing the test current by that at the end of the prepulse corrects for variation in the activation of the gH during different prepulses. The symbols have the same meaning as in A.
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Figure 2: (A) Instantaneous current–voltage relationships in a cell studied at pH 7.0//6.5 before (•) and after (▪) addition of 10 μM ZnCl2 to the bath. A prepulse to +40 mV for control and +100 mV in the presence of ZnCl2 was applied to open H+ channels, followed by a test pulse to the voltage on the abscissae. The current at the start of the test pulse, after the capacitive transient, is plotted. (B) Data from A after correction for the current at the end of the prepulse, and normalized to be equal at +100 mV. Dividing the test current by that at the end of the prepulse corrects for variation in the activation of the gH during different prepulses. The symbols have the same meaning as in A.

Mentions: If ZnCl2 binds with rapid kinetics to a site in the H+ channel within the membrane electrical field, this should manifest itself in the instantaneous current–voltage relationship. The control instantaneous current–voltage (I-V) relationship in Fig. 2 A (•) exhibits moderate outward rectification, consistent with previous studies (Byerly et al. 1984; Kapus et al. 1993; Bernheim et al. 1993; Cherny et al. 1995; DeCoursey and Cherny 1996). The instantaneous I-V relationship in the presence of 10 μM ZnCl2 (▪) is also plotted. The currents are reduced even though the prepulse was 40 mV more positive. After both sets of currents are scaled to match at +100 mV (Fig. 2 B), the currents superimpose, indicating that there is no rapid voltage-dependent block. In some experiments with CdCl2, there was a suggestion that the inward currents were reduced preferentially, but this effect was too small to be sure of, even with data spanning 200 mV. Thus, metals have negligible effects on the instantaneous I-V relation of H+ channels.


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)

(A) Instantaneous current–voltage relationships in a cell studied at pH 7.0//6.5 before (•) and after (▪) addition of 10 μM ZnCl2 to the bath. A prepulse to +40 mV for control and +100 mV in the presence of ZnCl2 was applied to open H+ channels, followed by a test pulse to the voltage on the abscissae. The current at the start of the test pulse, after the capacitive transient, is plotted. (B) Data from A after correction for the current at the end of the prepulse, and normalized to be equal at +100 mV. Dividing the test current by that at the end of the prepulse corrects for variation in the activation of the gH during different prepulses. The symbols have the same meaning as in A.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: (A) Instantaneous current–voltage relationships in a cell studied at pH 7.0//6.5 before (•) and after (▪) addition of 10 μM ZnCl2 to the bath. A prepulse to +40 mV for control and +100 mV in the presence of ZnCl2 was applied to open H+ channels, followed by a test pulse to the voltage on the abscissae. The current at the start of the test pulse, after the capacitive transient, is plotted. (B) Data from A after correction for the current at the end of the prepulse, and normalized to be equal at +100 mV. Dividing the test current by that at the end of the prepulse corrects for variation in the activation of the gH during different prepulses. The symbols have the same meaning as in A.
Mentions: If ZnCl2 binds with rapid kinetics to a site in the H+ channel within the membrane electrical field, this should manifest itself in the instantaneous current–voltage relationship. The control instantaneous current–voltage (I-V) relationship in Fig. 2 A (•) exhibits moderate outward rectification, consistent with previous studies (Byerly et al. 1984; Kapus et al. 1993; Bernheim et al. 1993; Cherny et al. 1995; DeCoursey and Cherny 1996). The instantaneous I-V relationship in the presence of 10 μM ZnCl2 (▪) is also plotted. The currents are reduced even though the prepulse was 40 mV more positive. After both sets of currents are scaled to match at +100 mV (Fig. 2 B), the currents superimpose, indicating that there is no rapid voltage-dependent block. In some experiments with CdCl2, there was a suggestion that the inward currents were reduced preferentially, but this effect was too small to be sure of, even with data spanning 200 mV. Thus, metals have negligible effects on the instantaneous I-V relation of H+ channels.

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