Voltage-activated proton current in eosinophils from human blood.
1. The resting membrane potential of freshly purified normodense human eosinophils bathed in and dialysed with quasi-physiological solutions was -63 +/- 2 mV (n = 100). 2. In voltage-clamp mode with quasi-physiological internal and external solutions, voltage steps from the holding potential of -60 mV to levels positive to +20 mV resulted in development of a quasi-instantaneous outward current and a slowly developing outward current. The instantaneous current was absent when the cells were bathed in and dialysed with K(+)-free solution. 3. The slow outward current persisted following simultaneous replacement of K+, Na+ and most of the Cl- with largely impermeant ions (tetraethylammonium, N-methyl-D-glucamine and methanesulphonate) and was augmented when the cell was dialysed with a solution of increased buffering capacity for protons. The observed reversal potential of the current closely followed the hydrogen equilibrium potential over a wide range of internal-external pH combinations, indicating that the conductance underlying the slow outward current was highly selective for H+ ions. 4. Acidification of the pipette solution (increasing [H+]i) augmented the outward H+ current and shifted its activation range negatively, whilst acidification of the external solution had the opposite effect. The voltage dependence of the current is modulated by the transmembrane pH gradient so the only outward current could be activated. However, when the outward current was activated by a voltage step, rapid acidification of external solution produced an inward H+ current which rapidly deactivated. 5. The proton current was reversibly inhibited in a voltage-dependent manner by extracellular application of Zn2+. The apparent dissociation constants were 8 nM (at +40 mV), 36 nM (at +70 mV) and 200 nM (at +100 mV). 6. The proton current was augmented by exposure to 10 microM arachidonic acid. This augmentation consisted of a shift of the voltage dependence of activation to more negative potentials and enhancement of maximum conductance (gH,max). The proton current recorded in eosinophils was significantly augmented under conditions of elevated cytosolic free calcium concentration ([Ca2+]i). The threshold level of [Ca2+]i associated with this effect lay between 0.1 and 1 microM and was not measurably affected by cytosolic acidification. 7. Eosinophils from human blood possess a voltage-dependent H+ conductance (gH) which normally allows protons to move outwards only; raising [Ca2+]i was associated with augmentation of gH and intracellular acidification or arachidonate shifted its activation range negatively towards physiological potentials.
Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London, UK.
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DV
Gordienko
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Tare
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Parveen
C J
CJ
Fenech
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Robinson
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Bolton
D000328:Adult; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D004594:Electrophysiology; D004804:Eosinophils / Q000378:metabolism*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008875:Middle Aged; D018408:Patch-Clamp Techniques; D011522:Protons; D015032:Zinc / Q000494:pharmacology
D007473:Ion Channels; D011522:Protons; D016718:Arachidonic Acid; D015032:Zinc; D002118:Calcium