Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Effects of ZnCl2 on the H+ current elicited by a 4-s pulse to +10 mV in a cell studied at pH 7.0//5.5. The inset shows the pH of the pipette and bath solutions. (B) The same currents scaled to the same value at the start and end of the 4-s pulse, illustrating the slowing of the activation time course. The steps in the 10 μM record are due to the resolution of the A–D converter.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230650&req=5

Figure 1: (A) Effects of ZnCl2 on the H+ current elicited by a 4-s pulse to +10 mV in a cell studied at pH 7.0//5.5. The inset shows the pH of the pipette and bath solutions. (B) The same currents scaled to the same value at the start and end of the 4-s pulse, illustrating the slowing of the activation time course. The steps in the 10 μM record are due to the resolution of the A–D converter.

Mentions: The inhibition of H+ currents by external ZnCl2 is illustrated in Fig. 1. The H+ current elicited by a pulse to +10 mV is reduced in a concentration-dependent manner by ZnCl2. The rate the current turns on during a depolarizing voltage pulse is slower, as seen more clearly in Fig. 1 B, where the currents are scaled to the same value at the end of the pulse. Another effect (explored below) is to shift the voltage dependence of H+ channel gating to more positive voltages. To some extent, the reduced H+ current amplitude and slower activation can be attributed to this voltage shift. One implication is that any attempt to quantitate the apparent “block” of H+ currents by ZnCl2 by measuring the current at the end of a pulse will be arbitrary because the result depends strongly on the length of the pulse and the voltage selected for the measurement. The apparent extent of block at the end of the pulses in Fig. 1 would be greatly reduced if longer test pulses were applied and especially if a more positive test potential were selected.


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) Effects of ZnCl2 on the H+ current elicited by a 4-s pulse to +10 mV in a cell studied at pH 7.0//5.5. The inset shows the pH of the pipette and bath solutions. (B) The same currents scaled to the same value at the start and end of the 4-s pulse, illustrating the slowing of the activation time course. The steps in the 10 μM record are due to the resolution of the A–D converter.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) Effects of ZnCl2 on the H+ current elicited by a 4-s pulse to +10 mV in a cell studied at pH 7.0//5.5. The inset shows the pH of the pipette and bath solutions. (B) The same currents scaled to the same value at the start and end of the 4-s pulse, illustrating the slowing of the activation time course. The steps in the 10 μM record are due to the resolution of the A–D converter.
Mentions: The inhibition of H+ currents by external ZnCl2 is illustrated in Fig. 1. The H+ current elicited by a pulse to +10 mV is reduced in a concentration-dependent manner by ZnCl2. The rate the current turns on during a depolarizing voltage pulse is slower, as seen more clearly in Fig. 1 B, where the currents are scaled to the same value at the end of the pulse. Another effect (explored below) is to shift the voltage dependence of H+ channel gating to more positive voltages. To some extent, the reduced H+ current amplitude and slower activation can be attributed to this voltage shift. One implication is that any attempt to quantitate the apparent “block” of H+ currents by ZnCl2 by measuring the current at the end of a pulse will be arbitrary because the result depends strongly on the length of the pulse and the voltage selected for the measurement. The apparent extent of block at the end of the pulses in Fig. 1 would be greatly reduced if longer test pulses were applied and especially if a more positive test potential were selected.

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