The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

Vladimir V. Cherny, Vladislav S. Markin, Thomas E. Decoursey

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Abstract

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, V(rev), was close to the Nernst potential, E(H), varying 52 mV/U pH over four ΔpH units (ΔpH = pH(o) - pH(i)). This result indicates that H+ channels are extremely selective, P(H)/P(TMA) > 107, and that both internal and external pH, pH(i), and pH(o), were well controlled. The H+ current amplitude was practically constant at any fixed ΔpH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current-voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, g(H), as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by ~40 mV/U pH by changes in phi or pH(o), with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as ~20-40(pH(o)-pH(i)) mV. If internal and external protons regulate the voltage dependence of g(H) gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, ΔpH. We propose a simple general model to account for the observed ΔpH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on ΔpH and not on the absolute pH.

Original languageEnglish (US)
Pages (from-to)861-896
Number of pages36
JournalJournal of General Physiology
Volume105
Issue number6
DOIs
StatePublished - Jun 1995

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Alveolar Epithelial Cells
Proton-Motive Force
Protons
Tail

ASJC Scopus subject areas

  • Physiology

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The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. / Cherny, Vladimir V.; Markin, Vladislav S.; Decoursey, Thomas E.

In: Journal of General Physiology, Vol. 105, No. 6, 06.1995, p. 861-896.

Research output: Contribution to journalArticle

Cherny, Vladimir V. ; Markin, Vladislav S. ; Decoursey, Thomas E. / The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. In: Journal of General Physiology. 1995 ; Vol. 105, No. 6. pp. 861-896.
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abstract = "Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, V(rev), was close to the Nernst potential, E(H), varying 52 mV/U pH over four ΔpH units (ΔpH = pH(o) - pH(i)). This result indicates that H+ channels are extremely selective, P(H)/P(TMA) > 107, and that both internal and external pH, pH(i), and pH(o), were well controlled. The H+ current amplitude was practically constant at any fixed ΔpH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current-voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, g(H), as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by ~40 mV/U pH by changes in phi or pH(o), with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as ~20-40(pH(o)-pH(i)) mV. If internal and external protons regulate the voltage dependence of g(H) gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, ΔpH. We propose a simple general model to account for the observed ΔpH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on ΔpH and not on the absolute pH.",
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N2 - Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, V(rev), was close to the Nernst potential, E(H), varying 52 mV/U pH over four ΔpH units (ΔpH = pH(o) - pH(i)). This result indicates that H+ channels are extremely selective, P(H)/P(TMA) > 107, and that both internal and external pH, pH(i), and pH(o), were well controlled. The H+ current amplitude was practically constant at any fixed ΔpH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current-voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, g(H), as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by ~40 mV/U pH by changes in phi or pH(o), with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as ~20-40(pH(o)-pH(i)) mV. If internal and external protons regulate the voltage dependence of g(H) gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, ΔpH. We propose a simple general model to account for the observed ΔpH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on ΔpH and not on the absolute pH.

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