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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) Slowing of τact by ZnCl2 depends strongly on pHo (▿, pHo 8; •, ○, 7; ▪, □, 6.0; ♦, 5.5; ▴, pHo 5) but is independent of pHi. Open symbols indicate cells studied at pHi 6.5, solid symbols at pHi 5.5. Families of H+ currents were recorded in the absence and presence of ZnCl2 in each cell. The H+ currents were fitted by a single exponential after a delay and the τact data plotted versus voltage, as illustrated in Fig. 4. The ratio of τact in the presence of ZnCl2 to that in its absence was measured at several voltages and averaged for each cell. When the voltage range did not overlap (as occurred for only a few cells at high [ZnCl2]), the control value was extrapolated from τact data at the highest voltages studied. The mean ratios from three to five different cells at each pHo//pHi are plotted along with SD bars. The dashed line indicates a ratio of 1.0, which means that no effect was observed. (B) The data in A are replotted after correcting for measured metal binding by the buffers used (Table ). The corrections apply to measurements using PIPES and Mes, for which detectable binding of ZnCl2 was measured. The calculated correction factors that give the fraction of total applied [ZnCl2] that is unbound by buffer are: 0.576 for pHo 7.0, 0.798 for pHo 6.0, and 0.90 for pHo 5.5, calculated from [M]free/[M]total = 1/(1 + K ′M[B−]), where the deprotonated buffer concentration [B−] was calculated by the Henderson-Hasselbalch equation according to the buffer pKa and pH. No correction was applied at pHo 8.0 or 5.0 because no binding of ZnCl2 to HEPES or Homopipes, respectively, was detected (Table ).
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Figure 6: (A) Slowing of τact by ZnCl2 depends strongly on pHo (▿, pHo 8; •, ○, 7; ▪, □, 6.0; ♦, 5.5; ▴, pHo 5) but is independent of pHi. Open symbols indicate cells studied at pHi 6.5, solid symbols at pHi 5.5. Families of H+ currents were recorded in the absence and presence of ZnCl2 in each cell. The H+ currents were fitted by a single exponential after a delay and the τact data plotted versus voltage, as illustrated in Fig. 4. The ratio of τact in the presence of ZnCl2 to that in its absence was measured at several voltages and averaged for each cell. When the voltage range did not overlap (as occurred for only a few cells at high [ZnCl2]), the control value was extrapolated from τact data at the highest voltages studied. The mean ratios from three to five different cells at each pHo//pHi are plotted along with SD bars. The dashed line indicates a ratio of 1.0, which means that no effect was observed. (B) The data in A are replotted after correcting for measured metal binding by the buffers used (Table ). The corrections apply to measurements using PIPES and Mes, for which detectable binding of ZnCl2 was measured. The calculated correction factors that give the fraction of total applied [ZnCl2] that is unbound by buffer are: 0.576 for pHo 7.0, 0.798 for pHo 6.0, and 0.90 for pHo 5.5, calculated from [M]free/[M]total = 1/(1 + K ′M[B−]), where the deprotonated buffer concentration [B−] was calculated by the Henderson-Hasselbalch equation according to the buffer pKa and pH. No correction was applied at pHo 8.0 or 5.0 because no binding of ZnCl2 to HEPES or Homopipes, respectively, was detected (Table ).

Mentions: Fig. 5 illustrates the effects of ZnCl2 on H+ currents at three pHo. ZnCl2 reduces the H+ current at each voltage, slows activation, and shifts the voltage dependence of activation to more positive voltages. At each pHo, the effects are similar, but the concentration of ZnCl2 required to produce these effects is much greater at low pHo. In this sense, lowering pHo decreases the efficacy of ZnCl2. To quantitate the effects of ZnCl2, we measured τact and calculated the ratio of τact in the presence of ZnCl2 to that in its absence in the same cell at the same voltage. In most cells, this ratio was the same at all voltages, thus the effect of ZnCl2 is a uniform voltage-independent slowing. Average ratios at several pHo are plotted in Fig. 6 and can be thought of as reflecting the “apparent potency” of ZnCl2 at various pHo. The concentration required to slow τact twofold is (μM) 0.22 at pHo 8, 0.46 at pHo 7, 5.4 at pHo 6, 89 at pHo 5.5, and 1,000 at pHo 5. The apparent potency of ZnCl2 (estimated for a fourfold slowing of τact where the curves are parallel) decreased only 2.3-fold between pHo 8 and 7, 10-fold between pHo 7 and 6, and 103-fold between pHo 6 and 5.


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) Slowing of τact by ZnCl2 depends strongly on pHo (▿, pHo 8; •, ○, 7; ▪, □, 6.0; ♦, 5.5; ▴, pHo 5) but is independent of pHi. Open symbols indicate cells studied at pHi 6.5, solid symbols at pHi 5.5. Families of H+ currents were recorded in the absence and presence of ZnCl2 in each cell. The H+ currents were fitted by a single exponential after a delay and the τact data plotted versus voltage, as illustrated in Fig. 4. The ratio of τact in the presence of ZnCl2 to that in its absence was measured at several voltages and averaged for each cell. When the voltage range did not overlap (as occurred for only a few cells at high [ZnCl2]), the control value was extrapolated from τact data at the highest voltages studied. The mean ratios from three to five different cells at each pHo//pHi are plotted along with SD bars. The dashed line indicates a ratio of 1.0, which means that no effect was observed. (B) The data in A are replotted after correcting for measured metal binding by the buffers used (Table ). The corrections apply to measurements using PIPES and Mes, for which detectable binding of ZnCl2 was measured. The calculated correction factors that give the fraction of total applied [ZnCl2] that is unbound by buffer are: 0.576 for pHo 7.0, 0.798 for pHo 6.0, and 0.90 for pHo 5.5, calculated from [M]free/[M]total = 1/(1 + K ′M[B−]), where the deprotonated buffer concentration [B−] was calculated by the Henderson-Hasselbalch equation according to the buffer pKa and pH. No correction was applied at pHo 8.0 or 5.0 because no binding of ZnCl2 to HEPES or Homopipes, respectively, was detected (Table ).
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Figure 6: (A) Slowing of τact by ZnCl2 depends strongly on pHo (▿, pHo 8; •, ○, 7; ▪, □, 6.0; ♦, 5.5; ▴, pHo 5) but is independent of pHi. Open symbols indicate cells studied at pHi 6.5, solid symbols at pHi 5.5. Families of H+ currents were recorded in the absence and presence of ZnCl2 in each cell. The H+ currents were fitted by a single exponential after a delay and the τact data plotted versus voltage, as illustrated in Fig. 4. The ratio of τact in the presence of ZnCl2 to that in its absence was measured at several voltages and averaged for each cell. When the voltage range did not overlap (as occurred for only a few cells at high [ZnCl2]), the control value was extrapolated from τact data at the highest voltages studied. The mean ratios from three to five different cells at each pHo//pHi are plotted along with SD bars. The dashed line indicates a ratio of 1.0, which means that no effect was observed. (B) The data in A are replotted after correcting for measured metal binding by the buffers used (Table ). The corrections apply to measurements using PIPES and Mes, for which detectable binding of ZnCl2 was measured. The calculated correction factors that give the fraction of total applied [ZnCl2] that is unbound by buffer are: 0.576 for pHo 7.0, 0.798 for pHo 6.0, and 0.90 for pHo 5.5, calculated from [M]free/[M]total = 1/(1 + K ′M[B−]), where the deprotonated buffer concentration [B−] was calculated by the Henderson-Hasselbalch equation according to the buffer pKa and pH. No correction was applied at pHo 8.0 or 5.0 because no binding of ZnCl2 to HEPES or Homopipes, respectively, was detected (Table ).
Mentions: Fig. 5 illustrates the effects of ZnCl2 on H+ currents at three pHo. ZnCl2 reduces the H+ current at each voltage, slows activation, and shifts the voltage dependence of activation to more positive voltages. At each pHo, the effects are similar, but the concentration of ZnCl2 required to produce these effects is much greater at low pHo. In this sense, lowering pHo decreases the efficacy of ZnCl2. To quantitate the effects of ZnCl2, we measured τact and calculated the ratio of τact in the presence of ZnCl2 to that in its absence in the same cell at the same voltage. In most cells, this ratio was the same at all voltages, thus the effect of ZnCl2 is a uniform voltage-independent slowing. Average ratios at several pHo are plotted in Fig. 6 and can be thought of as reflecting the “apparent potency” of ZnCl2 at various pHo. The concentration required to slow τact twofold is (μM) 0.22 at pHo 8, 0.46 at pHo 7, 5.4 at pHo 6, 89 at pHo 5.5, and 1,000 at pHo 5. The apparent potency of ZnCl2 (estimated for a fourfold slowing of τact where the curves are parallel) decreased only 2.3-fold between pHo 8 and 7, 10-fold between pHo 7 and 6, and 103-fold between pHo 6 and 5.

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