Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Abstract

Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH i in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na ؉ /H ؉ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH i decreased sharply, and recovery required both Na ؉ /H ؉ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn 2؉ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn 2؉ . Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis. innate immunity ͉ ion channels ͉ phagocyte ͉ respiratory burst ͉ SNARF W e have reinvestigated the regulation of cytoplasmic pH, pH i , in human neutrophils during phagocytosis. For 3 decades, pH i during the respiratory burst has been characterized as a small (0.05-0.1 unit) transient drop in pH i that is followed by larger (0.16-0.60 unit) prolonged alkalinization caused by Na ϩ /H ϩ antiport activity, whether the stimulus is fMLF (fMetLeuPhe, a chemotactic peptide), PMA (a PKC-activating phorbol ester), phospholipase C, or OPZ (opsonized zymosan) (1-7). In most studies, soluble stimuli were applied to populations of neutrophils. When phagocytosis was studied, the responses of many cells were averaged (5, 8). A powerful new imaging approach, confocal microscopy using the fluorescent pH indicator SNARF-1 enhanced by shifted excitation and emission ratioing (SEER) (9, 10) enabled us to examine the responses of individual cells with high spatial and temporal resolution. We report here that the behavior of individual human neutrophils during phagocytosis differs markedly from the prevailing view. The fundamental pH i response is triphasic: rapid and profound acidification followed by rapid but variably complete recovery, and after longer times, slow acidification that may reflect the onset of apoptosis. ResultsCells That Phagocytose Become Acidic. Human neutrophils were allowed to adhere to a g...

The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H+ channel Hv1

“…2a,b). In fact, a Hv channel exhibiting high temperature dependence 37,38 is expressed in phagocytes and lymphocytes and contributes to immunological events such as phagocytosis and the antibody production that occur under conditions of local and whole-body fever [3][4][5][6] . The temperature-dependent activation of mHv1/VSOP showed two key features in our study: the temperature dependences for activation (slopes of the channel gating) with the coiled-coil domain deleted (Fig.…”

“…H v1/VSOP is a recently discovered voltage-gated H + channel (Hv) 1,2 that is expressed in immunocytes and is involved in the production of reactive oxygen species in phagocytes [3][4][5] and the antigen response in B lymphocytes 6 . Hv1/VSOP has fourtransmembrane segments that correspond to the voltage-sensor domain (S1-S4) of other voltage-gated channels 1,2 , it does not have a canonical pore domain, and the voltage-sensor domain is thought to act as both the voltage-sensor and the H + -permeation pathway 7 .…”

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

“…The BKCa channel paper was eventually retracted (Retraction, 2010) and the cloning in 2006 of the HVCN1 gene coding for the Hv1 proton channel (Ramsey et al, 2006;Sasaki et al, 2006) enabled to test the role of Hv1 channels in antibacterial defense. Studies from mice lacking Hv1 proton channels (Ramsey et al, 2009;Morgan et al, 2009;El Chemaly et al, 2010) clearly established that Hv1 proton channels provide most of the charge compensation during the respiratory burst (see the Fig. 1).…”

“…All attempts to record proton currents in Hv1-deficient phagocytes yielded absolutely flat traces, even after activation of cells with PMA (Ramsey et al, 2009;Morgan et al, 2009;El Chemaly et al, 2010;Okochi et al, 2009), a procedure that evoked electron currents of similar amplitude in Hv1 -/-and in wild-type (WT) Fig. 1.…”

“…The mechanism outlined above whereby Hv1 proton channels sustain the activity of the oxidase NOX2 was derived from measurements performed at the whole-cell level, by measuring ROS production from cells activated with phorbol esters or with solid particles (Ramsey et al, 2009;Morgan et al, 2009;El Chemaly et al, 2010). In these conditions, it is impossible to tell whether the ROS are produced at the plasma membrane or in intracellular membranes.…”