Voltage‐gated proton channels (Hv1/VSOP) are expressed in various cells types, including phagocytes, and are involved in diverse physiological processes. Although hvcn1, the gene encoding Hv1, has been identified across a wide range of species, most of the knowledge about its physiological function and expression profile is limited to mammals. In this study, we investigated the basic properties of DrHv1, the Hv1 ortholog in zebrafish (Danio rerio) which is an excellent animal model owing to the transparency, as well as its functional expression in native cells. Electrophysiological analysis using a heterologous expression system confirmed the properties of a voltage‐gated proton channel are conserved in DrHv1 with differences in threshold and activation kinetics as compared to mouse (Mus musculus) Hv1 (mHv1). RT‐PCR analysis revealed that hvcn1 is expressed in zebrafish neutrophils, as is the case in mammals. Subsequent electrophysiological analysis confirmed the functional expression of DrHv1 in zebrafish neutrophils, which suggests Hv1 function in phagocytes is conserved among vertebrates. We also found that DrHv1 is comparatively resistant to extracellular Zn2+, which is a potent inhibitor of mammalian Hv1, and this phenomenon appears to reflect variation in the Zn2+‐coordinating residue (histidine) within the extracellular linker region in mammalian Hv1. Notably, the serum Zn2+ concentration is much higher in zebrafish than in mouse, raising the possibility that Zn2+ sensitivity was acquired in accordance with a change in the serum Zn2+ concentration. This study highlights the biological variation and importance of Hv1 in different animal species.
Voltage-sensing phosphatase (Vsp) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. Vsp orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of Vsp in native tissues remains largely unknown. Here we report that zebrafish Vsp (Dr-Vsp), encoded by tpte gene, is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-Vsp-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-Vsp-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis Vsp (Ci-Vsp) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of Vsp in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species.
Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos ( Danio rerio). Hvcn1-deficiency ( hvcn1−/−) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, hvcn1-deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and hvcn1−/− zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1−/− neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.
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