A primary function of the H+-ATPase (or V-ATPase) is to create an electrochemical proton gradient across eukaryotic cell membranes, which energizes fundamental cellular processes. Its activity notably allows for the acidification of intracellular vesicles and organelles, which is necessary for many essential cell biological events to occur. In addition, many specialized cell types in various organ systems such as the kidney, bone, male reproductive tract, inner ear, olfactory mucosa, and more, use plasma membrane V-ATPases to perform specific activities that depend on extracellular acidification. However, it is increasingly apparent that V-ATPases are central players in many normal and pathophysiological processes that directly influence human health in many different, and sometimes unexpected ways. These include cancer, neurodegenerative diseases, diabetes, and sensory perception, as well as energy and nutrient sensing functions within cells. This review first covers the well-established role of the V-ATPase as a transmembrane proton pump in the plasma membrane and intracellular vesicles, and outlines factors contributing to its physiological regulation in different cell types. This is followed by a discussion of the more recently emerging unconventional roles for the V-ATPase, such as its role as a protein interaction hub involved in cell signaling, and the (patho)physiological implications of these interactions. Finally, the central importance of endosomal acidification and V-ATPase activity on viral infection will be discussed in the context of the current COVID-19 pandemic.

Changes in the urothelial barrier are observed in patients with cystitis, but whether this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 vs. controls transduced with green fluorescent protein. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.

A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na channels (ENaC), but if all Na reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na over K, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down β- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.

The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKC␣ influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKC␣ Ϫ/Ϫ mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKC␣, the open probability (P o) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 Ϯ 0.04 vs. 0.17 Ϯ 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of ␣-, ␤-, and ␥-subunits of ENaC in principal cells in the cortical collecting ducts of PKC␣ Ϫ/Ϫ mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKC␣ Ϫ/Ϫ mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and P o by PKC␣ in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.protein kinase C␣; ENaC; renal tubules; single channels; knockout mice; hypertension EPITHELIAL NA CHANNELS (ENaC) are sodium-permeable ion channels located in the apical membrane of polarized epithelial cells primarily in the distal nephron, lung, and distal colon. In the distal nephron, ENaC activity is the rate-limiting step for Na ϩ reabsorption (16, 34); therefore, ENaC activity is critical for the physiological maintenance of systemic Na ϩ homeostasis and long-term control of blood pressure. Because of its central role in responding to changes in Na ϩ uptake, ENaC activity is tightly regulated; dysregulation of this channel has been linked to abnormal blood pressure in several genetic disorders including Liddle's syndrome (18, 37) and pseudohypoaldosteronism type 1 (9, 33, 41).ENaC can be regulated either by altering the amount of time the channel spends open (open probability or P o ) or by altering the density of functional channels (N) in the apical membrane of distal nephron epithelial cells. One signaling molecule that appears to alter ENaC activity is protein kinase C (PKC). Activation of PKC with phorbol esters reduces ENaC activity in the apical membrane of A6 cells, an amphibian renal cell line, and in rat principal cells (15). In contrast to the inhibitory effect on ENaC due to activating PKC, pharmacologically inhibiting PKC increases ENaC P o (23, 49). A6 cells, on which many of the experiments described above were performed, contain several different PKC isoforms; so that it is difficult to determine which isoform is responsible for the changes in E...