Properties of Single Voltage-gated Proton Channels in Human Eosinophils Estimated by Noise Analysis and by Direct Measurement

Abstract

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi ≤ 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, g H-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{H}}}^{{\mathrm{2}}}\end{equation*}\end{document}. We analyze \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{H}}}^{{\mathrm{2}}}\end{equation*}\end{document} data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic g H was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic g H, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi ≤ 5.5 near V threshold (the voltage at which g H turns on), single channel–like current events were observed with amplitudes 7–16 fA.

Philosophy of voltage-gated proton channels

“…The human proton channel hH V 1, has an estimated conductance of 78 fS at physiological pH and human body temperature [111]. Given a large driving force, say 100 mV, one channel would carry 7.8 fA of H þ current, which translates into 5 Â 10 4 H þ s 21 .…”

Analysis of Electrophysiological Properties and Responses of Neutrophils

“…Under optimal conditions, seals can reach the TΩ (10 12 Ω) range (37). This high resistance seal allows recordings with very little background noise and small leak currents (i.e., current that flows from the pipette into the bath through the seal, rather than through the membrane).…”

“…Recording small currents with the patch clamp technique requires a large signal-to-noise ratio. The smallest directly-recorded single-channel currents were ~8 fA proton channel currents in human eosinophils (37). Noise in the patch clamp set-up arises from the electronic circuitry, the pipette and holder assembly, the seal and cell membrane, and extraneous mechanical or electrical sources (36,75).…”

“…Proton channels have a conductance of ~15 fS at physiological pH (37), which corresponds with currents of a few femtoamperes, on the order of ~10,000 H + /s, depending on the driving voltage. Store-operated Ca 2+ (CRAC) channels may be present in neutrophils (57) and have similarly small conductance (58).…”

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils

“…Estimates of the rate of free proton generation indicate that catastrophic acidification would occur within the cell unless an efficient proton extruding mechanism was activated in parallel with electron transport (Rotstein et al, 1987). Several studies have shown that a voltage-dependent, Zn 2+ -sensitive plasma membrane proton channel is activated in parallel with the increase in NADPH oxidase activity Cherny et al, 2003;DeCoursey et al, 2001;DeCoursey et al, 2003;Gordienko et al, 1996;Petheo et al, 2003). This channel is regulated by mediators that increase PKCδ activity and, following PKC activation, the cytosolic pH (pH i ) does not significantly change in response to increases in superoxide production (Bankers-Fulbright et al, 2001).…”