Ph-Dependent Inhibition of Voltage-Gated H+ Currents in Rat Alveolar Epithelial Cells by Zn2+ and Other Divalent Cations

Self Cite

Eosinophils and other phagocytes use NADPH oxidase to kill bacteria. Proton channels in human eosinophils and neutrophils are thought to sustain NADPH oxidase activity, and their opening is greatly enhanced by a variety of NADPH oxidase activators, including phorbol myristate acetate (PMA). In nonphagocytic cells that lack NADPH oxidase, no clear effect of PMA on proton channels has been reported. The basophil is a granulocyte that is developmentally closely related to the eosinophil but nevertheless does not express NADPH oxidase. Thus, one might expect that stimulating basophils with PMA would not affect proton currents. However, stimulation of human basophils in perforated-patch configuration with PMA, N-formyl-methionyl-leucyl-phenylalanine, or anti-IgE greatly enhanced proton currents, the latter suggesting involvement of proton channels during activation of basophils by allergens through their highly expressed IgE receptor (Fc RI). The anti-IgE-stimulated response occurred in a fraction of cells that varied among donors and was less profound than that to PMA. PKC inhibition reversed the activation of proton channels, and the proton channel response to anti-IgE or PMA persisted in Ca 2؉ -free solutions. Zn 2؉ at concentrations that inhibit proton current inhibited histamine release elicited by PMA or anti-IgE. Studied with confocal microscopy by using SNARF-AM and the shifted excitation and emission ratioing of fluorescence approach, anti-IgE produced acidification that was exacerbated in the presence of 100 M Zn 2؉ . Evidently, proton channels are active in basophils during IgE-mediated responses and prevent excessive acidification, which may account for their role in histamine release.asthma ͉ histamine ͉ patch-clamp ͉ HNVCN1 ͉ allergy

Section: Anti-ige Responses Insupporting

confidence: 86%

Section: Anti-ige Responses Inmentioning

confidence: 93%

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils

Musset¹,

Morgan²,

Cherny³

et al. 2008

Self Cite

“…Another hallmark feature of Hv channels is zinc-sensitivity (26,28,29). Consistent with that feature, outward currents of A206stop were reversibly suppressed by 100 μM zinc in all cases (n = 3) (Fig.…”

Section: Resultssupporting

confidence: 68%

Functionality of the voltage-gated proton channel truncated in S4

Sakata

Kurokawa

Nørholm

et al. 2009

The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteinescanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.ion conduction | membrane topology | membrane insertion | voltage sensor V oltage sensor domain (VSD) is a protein module that senses transmembrane voltage and regulates ion permeation through the pore (1-3) and phosphoinositide turnover by the phosphatase (4). The fourth transmembrane segment (S4) of VSD contains periodically aligned positively charged residues (arginine and lysine), which play a critical role in voltage sensing (1, 2). Recent analyses of the crystal structures of voltage-gated potassium channels (5, 6) showed that basic residues in S4 form salt bridges with negatively charged residues from other helices, and these salt bridges are formed at both sides of a hydrophobic residue such as phenylalanine. This suggests a focused electric field within a crevice surrounded by transmembrane helices (6), which would enable extracellular and intracellular aqueous environments to enter deeply into the membrane through a narrow crevice. One line of functional evidence for this narrow crevice is that mutation within the VSD creates an ion permeation pathway that is activated upon hyperpolarization (7−9). Such ion conduction is called an "omegacurrent" (9) or "gating pore current" (10). Similar conductances through the VSD are elicited in a mutated form of human voltagegated sodium channels (SCN4a) found in patients with periodic paralysis (11, 12) and in flatworm potassium channels (13).Recently, the protein, which consists solely of a VSD, was identified in tunicate, mouse (14) and human (15) and called voltage sensor only protein (VSOP) or Hv1. Mouse VSOP (mVSOP) is found within the phagosomes of white blood cells, and is essential for robust production of superoxide anions (16, 17) and maintenance of intracellular pH (18) in phagocytes. Despite the lack of a pore domain, these proteins function as voltage-gated proton channels (Hv channels) in heterologous expression systems (14,15) and when reconstituted into artificial membranes (19). S4 of both mVSOP and Hv1 contain three argini...

“…Type II alveolar epithelial cells were isolated from adult male Sprague-Dawley rats by using enzyme digestion, lectin agglutination, and differential adherence, and maintained in primary tissue culture, as described elsewhere (15). Standard patch-clamp techniques at 20°C, with modifications described below, and computerized data acquisition and analysis were used (15). For whole-cell recording, the bath and pipette solutions contained 100 mM buffer near its pK a , tetramethylammonium methanesulfonate to bring the osmolarity to Ϸ300 mOsm, 1 mM EGTA, and 2 mM MgCl 2 or CaCl 2 (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils

DeCoursey

Cherny

Zhou

et al. 2000

130

266

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this ''respiratory burst'' in human neutrophils. Although stoichiometric H ؉ efflux occurs during the respiratory burst, efforts to stimulate voltagegated H ؉ channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H ؉ current properties, each of which increases the H ؉ current at any given voltage: (i) a 40-mV negative shift in the H ؉ conductance-voltage (gH-V) relationship; (ii) faster activation [smaller activation time constant ( act)] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant ( tail)] on repolarization; and (iv) a larger maximum H ؉ conductance (gH,max). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of tail with a time course paralleling the inhibition of electron current. However, the amplitudes of H ؉ and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H ؉ channels rather than inducing a new H ؉ channel.respiratory burst ͉ hydrogen ion ͉ pH ͉ proton channel ͉ ion channels N ADPH oxidase is a major bactericidal weapon of neutrophils and other phagocytes (1). On stimulation, the enzyme complex assembles in the membrane and produces superoxide anion, the precursor to several bactericidal reactive oxygen species. NADPH oxidase is electrogenic (2), releasing a proton into the cytoplasm for each superoxide anion released into the phagocytic vacuole or extracellular medium. As a result, pH changes reflecting increased H ϩ permeability are seen during the respiratory burst in phagocytes (2, 3) after stimulation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Voltagegated proton channels, discovered in snail neurons (4), were postulated to mediate the increased H ϩ permeability during the respiratory burst in human neutrophils (2, 5, 6). The importance of H ϩ efflux as a means of charge compensation is emphasized by the inhibition of PMA-stimulated superoxide anion production by Zn 2ϩ or Cd 2ϩ , which presumably act by inhibiting proton current (7,8). However, attempts to demonstrate effects of PMA on H ϩ currents in phagocytes studied in the whole-cell patchclamp configuration have heretofore been unsuccessful. It is therefore unclear whether the g H observed in phagoc...