Localization of the V-ATPase-interacting protein, TBC1D24, in rodent kidney

The V-ATPase, or proton pump, generates electrochemical gradients across membranes driving cell biological processes. The kidney-specific V-ATPase is expressed in intercalated cells (ICs), which regulate acid-base homeostasis. In the kidney, A-IC transport protons into the tubule lumen while B-IC move protons into the blood. Recently, we identified a new class of V-ATPase-interacting proteins defined by their TLDc domain (NCOA7, OXR1, TBC1D24, TLDC1, and TLDC2), all of which interact with the IC-specific B1 subunit of the V-ATPase and are involved in the reversible assembly of the V-ATPase into a functional holoenzyme. Among the TLDc proteins, TBC1D24 stands out because more than 40 unique pathogenic variants are reported for this gene in humans primarily linked to neurological disorders, such as epilepsy and intellectual disability, but also causing a wide spectrum of pathologies, ranging from deafness to kidney disorders. Despite, the link between TBC1D24 mutations and disease the molecular mechanisms of TBC1D24 disorders are not well understood. This is in part due the fact that commercial antibodies for TBC1D24 have proven unreliable leaving the subcellular localization of the protein largely unexamined. Here, we developed a novel rabbit polyclonal antibody against a C-terminal sequence of human TBC1D24. By western blotting, the anti-TBC1D24 antibody specifically recognized a single band at the expected molecular weight of ~63 kDa in lysates from HEK293 cells stably transfected with TBC1D24 mRNA. Subsequently, we used this antibody to examine the localization of TBC1D24 in rat kidney using indirect immunofluorescence. Staining with specific antibodies against the ubiquitous B2 subunit isoform and the “kidney” IC-specific B1 subunit isoform of the V-ATPase, we found that TBC1D24 was most highly expressed in the A-IC in the inner medullary collecting ducts (CD), although there was also a weaker, diffuse cytoplasmic staining in adjacent principal cells (PC). TBC1D24 also localized throughout the cytoplasm in IC but was most concentrated at the apical pole. Interestingly, there was minimal localization of TBC1D24 in the B1 V-ATPase-rich apical microvilli. This is consistent with the involvement of TBC1D24 in the reversible assembly of the V-ATPase holoenzyme, because V-ATPase complexes within the microvilli are already fully assembled and functional. A low level of TBC1D24 expression was detectable in the descending thin limbs of Henle in the inner medulla. In the outer medulla TBC1D24 was expressed in CD, as well as the thick ascending limb of Henle where staining was concentrated in the apical domain. In the cortex, TBC1D24 was localized in distal convoluted tubules (DCT), connecting segments (CNT), and CD, but not in proximal tubules or glomeruli. In cortical CD TBC1D24 was still enriched in IC, as in the medulla, but the expression level was lower, and there was also a low level of expression in adjacent PC. However, in the cortex, TBC1D24 is more highly expressed in B-type IC. TBC1D24 showed diffuse cytoplasmic localization in all the cells of the DCT and CNT, except for the IC present in these sections, where TBC1D24 localized to the apical domain. Moving forward, we plan to examine the localization of TBC1D24 in the brain where mutations are linked to neurological disorders in humans and determine whether pathogenic mutations alter the localization of TBC1D24 in the brain or kidney. We will also analyze TBC1D24 subcellular localization by electron microscopy to provide detailed information on the localization of TBC1D24, thus providing clues about its cellular function and how this may be targeted for therapeutic intervention. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.