Voltage‐activated proton current in eosinophils from human blood.

Abstract

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+]1) 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 that 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 ,UM arachidonic acid. This augmentation consisted of a shift of the voltage dependence of activation to more negative potentials and enhancement of maximum conductance (H,max Assessment of the role of the eosinophil in health and disease eosinophils are now incriminated as important effector cells has proved difficult because these cells constitute only a in a number of diverse human diseases. These include the small percentage of the total leucocyte population. This host response to parasite infestation and tumours, the contrasts with the relatively sophisticated understanding reaction to transplanted tissues, and both allergic and nonthat exists for the more abundant neutrophil. Nevertheless, allergic inflammatory reactions of the major organ systems (reviewed by Spry, 1988).* To whom correspondence should be addressed. Receptor-directed stimulation of eosinophils and neutrophils leads to the release of a wide repertoire of chemical mediators. Some of these exist preformed within secretory granules, whilst others, such as eicosanoids and oxidants, are generated de novo when the cell is stimulated. Compared with neutrophils, there is a poor understanding of the events that...

Regulation of eosinophil membrane depolarization during NADPH oxidase activation

“…Although several groups have described the presence of proton channels in eosinophils (Gordienko et al, 1996;Schrenzel et al, 1996;Bánfi et al, 1999;Bankers-Fulbright et al, 2001;Cherny et al, 2001;DeCoursey et al, 2001a;DeCoursey et al, 2001b), the membrane potential determined by current clamp measurements indicated that depolarization following activation of NADPH oxidase was not sufficient to activate the proton channel (Gordienko et al, 1996;Tare et al, 1998;Bánfi et al, 1999). Eosinophil resting membrane potential has been reported to be between -60 and -80 mV (Gordienko et al, 1996;Tare et al, 1998). After activation of eosinophil NADPH oxidase with intracellular NADPH and GTPγS and after blocking the inwardly rectifying K + channel, Bánfi et al (Bánfi et al, 1999) reported a 40 mV depolarization.…”

“…Cytosolic protons are generated directly by NADPH oxidation and indirectly by regeneration of NADPH via the hexose monophosphate shunt (Borregaard et al, 1984;Lukacs et al, 1993). Associated with NADPH oxidase is an outwardly rectifying proton channel that is functionally required for continued oxidase activity (Henderson et al, 1988;Gordienko et al, 1996;Schrenzel et al, 1996). This NADPH oxidaseassociated proton channel is ligand-and voltage-regulated and has a predicted reversal potential that is nearly equal to the equilibrium potential for protons (EH).…”

“…Blocking the proton conductance with 250 µM ZnCl2 (+43.0±4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 neutrophils. Subsequently, current clamp recordings on eosinophils suggested an absolute resting membrane potential of approximately -60 to -80 mV (Gordienko, 1996;Tare, 1998), and an increase of 30-40 mV following NADPH oxidase activation with NADPH and GTPγS (Bánfi et al, 1999). Therefore, on the basis of current data describing eosinophil plasma membrane potential, the NADPH oxidaseassociated proton conductance is unlikely to be activated in eosinophils.…”

Analysis of Electrophysiological Properties and Responses of Neutrophils

“…The first patch clamp study on human granulocytes described nonselective cation channels activated by increased intracellular free calcium (3). Subsequently, the patch clamp technique has been used to explore proton, chloride, and potassium currents in neutrophils and eosinophils (4)(5)(6)(7)(8), as well as in many other nonexcitable cells.…”

“…Many studies have been done with ~100 mM buffer, and concentrations as high as 200 mM buffer with no other cations have been used (14) (see Note 6). Table 1 shows examples of solutions that have been used to study H + channels (22,6,28). …”