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Review
. 2002 Dec;120(6):759-65.
doi: 10.1085/jgp.20028708.

Proton conduction through gp91phox

Lydia M Henderson  1 , Robert W Meech
Affiliations
Review

Proton conduction through gp91phox

Lydia M Henderson et al. J Gen Physiol. 2002 Dec.
No abstract available

PubMed Disclaimer

Figures

F<sc>igure</sc> 1.
Figure 1.
Expression of NADPH Oxidase (Nox) protein in PLB and PLBKO cells. (A) Nox protein immunodetected in undifferentiated (i) and differentiated (ii) PLB-298 cells after incubation with an anti–COOH-terminal gp91phox polyclonal antibody and FITC-labeled anti–rabbit antibody. The polyclonal anti-gp91phox was raised to the COOH-terminal 14-amino acid peptide coupled to keyhole limpet hemocyanin. It was verified by Western blot against detergent-solubilized membrane proteins from human neutrophils (Henderson et al., 1995). The images, which show an optical slice close to the cell center, were obtained using a BioRad MRC 600 inverted confocal microscope. A pseudocolor scale in which the highest intensity of emitted fluorescence is denoted by red through orange, yellow and green to blue, low fluorescence, indicates the presence of a Nox protein. Differentiated PLB-985 cells treated in the absence of the anti-gp91phox antibody are shown in iii. (B) Immunodetection of Nox protein in PLBKO cells using the same technique and antibody as described for A. As before, undifferentiated (i), differentiated (ii), and control cells in the absence of anti-gp91phox antibody (iii) are shown. (C) Whole-cell currents recorded from a single differentiated PLB-985 cell under conditions designed to maximize the amplitude of H+ current (see Henderson and Meech, 1999). The patch pipette–filling solution, which was adjusted to pH 6.5, contained 119 mM TMA hydroxide, to block potassium currents, a small quantity of calcium buffer (3.7 mM EGTA, 0.74 mM CaCl2) and ∼120 mM MES pH buffer. The bathing solution contained 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, and 100 mM EPPS buffer adjusted to pH 8.0. Low amplitude prepulses in the linear current–voltage range (−60 to −100 mV) were scaled by PCLAMP 6 software (Axon Instruments, Inc.) and used for online subtraction of linear capacitive and leakage currents (Armstrong and Bezanilla, 1974). The superimposed currents shown were recorded in response to a standard stepped-voltage protocol; commands were in the range -40 to 40 mV (20-mV increments). The command duration was 100 ms (i) or 5 s (ii). The immunostaining of PLB-985 and PLBKO cells and recording of whole cell currents are the work of X. Wen Hu. Mary C. Dinauer provided the PLBKO cells. EPPS is N-[2-hydroxyethyl]-piperazine-N'-[3-propane-sulphonic acid]; TMA, tetramethylammonium.
F<sc>igure</sc> 1.
Figure 1.
Expression of NADPH Oxidase (Nox) protein in PLB and PLBKO cells. (A) Nox protein immunodetected in undifferentiated (i) and differentiated (ii) PLB-298 cells after incubation with an anti–COOH-terminal gp91phox polyclonal antibody and FITC-labeled anti–rabbit antibody. The polyclonal anti-gp91phox was raised to the COOH-terminal 14-amino acid peptide coupled to keyhole limpet hemocyanin. It was verified by Western blot against detergent-solubilized membrane proteins from human neutrophils (Henderson et al., 1995). The images, which show an optical slice close to the cell center, were obtained using a BioRad MRC 600 inverted confocal microscope. A pseudocolor scale in which the highest intensity of emitted fluorescence is denoted by red through orange, yellow and green to blue, low fluorescence, indicates the presence of a Nox protein. Differentiated PLB-985 cells treated in the absence of the anti-gp91phox antibody are shown in iii. (B) Immunodetection of Nox protein in PLBKO cells using the same technique and antibody as described for A. As before, undifferentiated (i), differentiated (ii), and control cells in the absence of anti-gp91phox antibody (iii) are shown. (C) Whole-cell currents recorded from a single differentiated PLB-985 cell under conditions designed to maximize the amplitude of H+ current (see Henderson and Meech, 1999). The patch pipette–filling solution, which was adjusted to pH 6.5, contained 119 mM TMA hydroxide, to block potassium currents, a small quantity of calcium buffer (3.7 mM EGTA, 0.74 mM CaCl2) and ∼120 mM MES pH buffer. The bathing solution contained 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, and 100 mM EPPS buffer adjusted to pH 8.0. Low amplitude prepulses in the linear current–voltage range (−60 to −100 mV) were scaled by PCLAMP 6 software (Axon Instruments, Inc.) and used for online subtraction of linear capacitive and leakage currents (Armstrong and Bezanilla, 1974). The superimposed currents shown were recorded in response to a standard stepped-voltage protocol; commands were in the range -40 to 40 mV (20-mV increments). The command duration was 100 ms (i) or 5 s (ii). The immunostaining of PLB-985 and PLBKO cells and recording of whole cell currents are the work of X. Wen Hu. Mary C. Dinauer provided the PLBKO cells. EPPS is N-[2-hydroxyethyl]-piperazine-N'-[3-propane-sulphonic acid]; TMA, tetramethylammonium.
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References

    1. Armstrong, C.M., and F. Bezanilla. 1974. Charge movement associated with the opening and closing of the activation gates of the Na channels. J. Gen. Physiol. 63:533–552. - PMC - PubMed
    1. Banfi, B., A. Maturana, S. Jaconi, S. Arnaudeau, T. Laforge, B. Sinha, E. Ligeti, N. Demaurex, and K.H. Krause. 2000. A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science. 287:138–142. - PubMed
    1. Banfi, B., G. Molnar, A. Maturana, K. Steger, B. Hegedus, N. Demaurex, and K.H. Krause. 2001. A Ca2+-activated NADPH oxidase in testis, spleen, and lymph nodes. J. Biol. Chem. 276:37594–37601. - PubMed
    1. Barish, M.E., and C. Baud. 1984. A voltage-gated hydrogen ion current in the oocyte membrane of the axolotl, Ambystoma. J. Physiol. 352:243–263. - PMC - PubMed
    1. Byerly, L., and S. Hagiwara. 1982. Calcium currents in internally perfused nerve cell bodies of Limnea stagnalis. J. Physiol. 322:503–528. - PMC - PubMed

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