Molecular and Functional Characterization of Hv1 Proton Channel in Human Granulocytes

Voltage-gated proton current (IHv) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of IHv in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently Hv1, a voltage-gated proton channel, was cloned, and leukocytes from Hv1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about Hv1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of Hv1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of Hv1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that Hv1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that Hv1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of Hv1 and Nox2 expression was observed in PLB-985 cells. The upregulation of Hv1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between Hv1 expression and IHv density in PLB-985 cells. It is also demonstrated that a massive reduction in Hv1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native Hv1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of Hv1 in granulocytes is optimized to support intense NADPH oxidase activity

The creation of a multiallele knockout genotype in rabbit using crispr/cas9 and its application in translational medicine

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase

The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O(2). The activity of the oxidase at the plasma membrane can be measured as electron current (I(e)), and the voltage dependence of I(e) was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from −120 to 200 mV, were applied to obtain ‘steady-state’ current–voltage relationships (I–V). The amplitude of I(e) recorded at −40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (K(m)) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of I(e) is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, I(e) was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (−60 to 60 mV), the I–V curve strongly rectifying only below −100 mV. At lower NADPH concentrations the I–V curve was progressively shifted to more positive potentials and I(e) became voltage-independent also within the physiological range. Consequently, the K(m) of the oxidase decreased by approx. 40% (from 100 to 60 μM) when the membrane potential increased from −60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I(e)–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I(e) reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase

Reactive oxygen species-mediated bacterial killing by B lymphocytes

Regulated production of ROS is mainly attributed to Nox family enzymes. In neutrophil granulocytes and macrophages, Nox2 has a crucial role in bacterial killing, and the absence of phagocytic ROS production leads to the development of CGD. Expression of Nox2 was also described in B lymphocytes, where the role of the enzyme is still poorly understood. Here, we show that peritoneal B cells, which were shown recently to possess phagocytic activity, have a high capacity to produce ROS in a Nox2-dependent manner. In phagocytosing B cells, intense intraphagosomal ROS production is detected. Finally, by studying 2 animal models of CGD, we demonstrate that phagocyte oxidase-deficient B cells have a reduced capacity to kill bacteria. Our observations extend the number of immune cell types that produce ROS to kill pathogens

Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase

The phagocytic NADPH oxidase (phox) moves electrons across cell membranes to kill microbes. The activity of this lethal enzyme is tightly regulated, but the mechanisms that control phox inactivation are poorly understood for lack of appropriate assays. The phox generates measurable electron currents, I(e), that are associated with inward proton currents, I(H). To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize I(e) (induced by the addition of NADPH) and I(H) (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. I(e) decayed rapidly in the absence of nucleotides (τ≈6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 μM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5′-[γ-thio]triphosphate) (τ≈57 min), but not by either ATP or GTP[S] alone. I(H) also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of I(H) required further addition of 25 μM PI(3,4)P(2) (phosphoinositide 3,4-bisphosphate) to the cytosolic side of the patch. PI(3,4)P(2) had no effect on I(e) and its stabilizing effect on I(H) could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased I(H) stability, an effect that was not prevented by phosphatase inhibitors but by the non-hydrolysable ATP analogue ATP[S] (adenosine 5′-[γ-thio]triphosphate). Our data indicate that the assembled phox complex is very stable in eosinophil membranes if both ATP and GTP[S] are present, but inactivates within minutes if one of the nucleotides is removed. Stabilization of the phox-associated proton channel in a highly voltage-sensitive conformation does not appear to involve phosphorylation but ATP binding, and requires not only ATP and GTP[S] but also PI(3,4)P(2), a protein known to anchor the cytosolic phox subunit p47(phox) to the plasma membrane