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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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Comparison of the τact data replotted from Fig. 6 with the slowing predicted by , assuming that the H+ channel cannot open while Zn2+ is bound to its receptor (see text for details). The meaning of the symbols is the same as in Fig. 6, and all curves are the predictions for pKa 6.3, pKM 6.5, and cooperativity factor a = 0.03.
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Figure 11: Comparison of the τact data replotted from Fig. 6 with the slowing predicted by , assuming that the H+ channel cannot open while Zn2+ is bound to its receptor (see text for details). The meaning of the symbols is the same as in Fig. 6, and all curves are the predictions for pKa 6.3, pKM 6.5, and cooperativity factor a = 0.03.

Mentions: Even though there is no rapidly reversible block, the more obvious effects of ZnCl2 could be due to a slow time-dependent block/unblock. Five arguments oppose the idea that the slow activation of H+ current in the presence of Zn2+ reflects voltage-dependent unbinding of Zn2+ from the channel. (a) If τact in the presence of metals (several seconds) reflects the unblock rate, then block must have very slow kinetics. If we assume that pKM = 6.5 (Fig. 11) and that the binding rate of Zn2+ is 3 × 107 M−1 s−1, a characteristic rate of complex formation between Zn2+ and proteins (Eigen and Hammes 1963), then the unbinding rate is 9.5 s−1. Thus, Zn2+ probably binds and unbinds in a fraction of a second. If the kinetics are rapid, effects should have been manifested in the instantaneous I-V relation. (b) In normal drug-receptor reactions, the unblock rate is independent of concentration. However, increasing the concentration of ZnCl2 slowed H+ current activation progressively. There was no indication that two populations of gating behavior resulted, as would be predicted if ZnCl2 modified a fraction of channels that then opened slowly, with the remaining channels opening at the normal rate. A single exponential (after a delay) continued to fit the data at all [ZnCl2]. Thus it appears that ZnCl2 binds and unbinds the channel repeatedly during a single pulse, with the slowing effect related to the fraction of time ZnCl2 is bound to the channel. (c) The steady state voltage dependence of this apparent Zn2+ block, defined as the ratio IH(Zn2+)/IH(control), is quite steep: a simple Boltzmann fit gives slope factors 8–13 mV (Fig. 3 D). In terms of traditional voltage-dependent block mechanisms (Woodhull 1973), if z is the charge on the blocking ion and δ is the fraction of the membrane potential sensed by the ion at the block site, then zδ ≥ 2.0, which implies that Zn2+, Cd2+, and Ni2+ traverse ≥100% of the membrane field to reach the block site. Several examples of δ > 1.0 for ionic blockade exist in the K+ channel literature and are traditionally explained by interaction between permeant ions in a multiply occupied channel (e.g., Hille and Schwarz 1978). Because it is unlikely for a hydrogen-bonded–chain conduction mechanism to support multiple protons simultaneously, especially at physiological pH (DeCoursey and Cherny 1999a), explaining the high zδ observed for divalent cation “blockade” is problematic. (d) If ZnCl2 simply shifted the gH-V relationship along the voltage axis, then the apparent steepness of the block, defined as the ratio IH(Zn2+)/IH(control), will be precisely identical to the steepness of the gH-V relationship in the absence of Zn2+. The slopes of the fractional block curves, 8–13 mV (Fig. 3D), and control gH-V relationships, 8–10 mV (DeCoursey and Cherny 1994; Cherny et al. 1995), are the same, consistent with a simple voltage shift. (e) Finally, any part of the H+ channel conductance pathway comprised of hydrogen-bonded chain would not allow Zn2+ passage; thus the possibility for voltage-dependent block by Zn2+ could exist only in an aqueous vestibule. We conclude that polyvalent cations do not exert their effects by entering into the pore, but instead bind to sites on the channel that are accessible to the solution and outside of the membrane potential field. Binding must be specific because different divalent cations have very different concentration dependencies. For example, effects of micromolar concentrations of Zn2+ are seen in the presence of millimolar [Ca2+]o or [Mg2+]o.


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)

Comparison of the τact data replotted from Fig. 6 with the slowing predicted by , assuming that the H+ channel cannot open while Zn2+ is bound to its receptor (see text for details). The meaning of the symbols is the same as in Fig. 6, and all curves are the predictions for pKa 6.3, pKM 6.5, and cooperativity factor a = 0.03.
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Related In: Results  -  Collection

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

Figure 11: Comparison of the τact data replotted from Fig. 6 with the slowing predicted by , assuming that the H+ channel cannot open while Zn2+ is bound to its receptor (see text for details). The meaning of the symbols is the same as in Fig. 6, and all curves are the predictions for pKa 6.3, pKM 6.5, and cooperativity factor a = 0.03.
Mentions: Even though there is no rapidly reversible block, the more obvious effects of ZnCl2 could be due to a slow time-dependent block/unblock. Five arguments oppose the idea that the slow activation of H+ current in the presence of Zn2+ reflects voltage-dependent unbinding of Zn2+ from the channel. (a) If τact in the presence of metals (several seconds) reflects the unblock rate, then block must have very slow kinetics. If we assume that pKM = 6.5 (Fig. 11) and that the binding rate of Zn2+ is 3 × 107 M−1 s−1, a characteristic rate of complex formation between Zn2+ and proteins (Eigen and Hammes 1963), then the unbinding rate is 9.5 s−1. Thus, Zn2+ probably binds and unbinds in a fraction of a second. If the kinetics are rapid, effects should have been manifested in the instantaneous I-V relation. (b) In normal drug-receptor reactions, the unblock rate is independent of concentration. However, increasing the concentration of ZnCl2 slowed H+ current activation progressively. There was no indication that two populations of gating behavior resulted, as would be predicted if ZnCl2 modified a fraction of channels that then opened slowly, with the remaining channels opening at the normal rate. A single exponential (after a delay) continued to fit the data at all [ZnCl2]. Thus it appears that ZnCl2 binds and unbinds the channel repeatedly during a single pulse, with the slowing effect related to the fraction of time ZnCl2 is bound to the channel. (c) The steady state voltage dependence of this apparent Zn2+ block, defined as the ratio IH(Zn2+)/IH(control), is quite steep: a simple Boltzmann fit gives slope factors 8–13 mV (Fig. 3 D). In terms of traditional voltage-dependent block mechanisms (Woodhull 1973), if z is the charge on the blocking ion and δ is the fraction of the membrane potential sensed by the ion at the block site, then zδ ≥ 2.0, which implies that Zn2+, Cd2+, and Ni2+ traverse ≥100% of the membrane field to reach the block site. Several examples of δ > 1.0 for ionic blockade exist in the K+ channel literature and are traditionally explained by interaction between permeant ions in a multiply occupied channel (e.g., Hille and Schwarz 1978). Because it is unlikely for a hydrogen-bonded–chain conduction mechanism to support multiple protons simultaneously, especially at physiological pH (DeCoursey and Cherny 1999a), explaining the high zδ observed for divalent cation “blockade” is problematic. (d) If ZnCl2 simply shifted the gH-V relationship along the voltage axis, then the apparent steepness of the block, defined as the ratio IH(Zn2+)/IH(control), will be precisely identical to the steepness of the gH-V relationship in the absence of Zn2+. The slopes of the fractional block curves, 8–13 mV (Fig. 3D), and control gH-V relationships, 8–10 mV (DeCoursey and Cherny 1994; Cherny et al. 1995), are the same, consistent with a simple voltage shift. (e) Finally, any part of the H+ channel conductance pathway comprised of hydrogen-bonded chain would not allow Zn2+ passage; thus the possibility for voltage-dependent block by Zn2+ could exist only in an aqueous vestibule. We conclude that polyvalent cations do not exert their effects by entering into the pore, but instead bind to sites on the channel that are accessible to the solution and outside of the membrane potential field. Binding must be specific because different divalent cations have very different concentration dependencies. For example, effects of micromolar concentrations of Zn2+ are seen in the presence of millimolar [Ca2+]o or [Mg2+]o.

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