Shushi Nagamori

This study identifies Na ⁺ /H ⁺ exchanger 3 (NHE3) as a key regulator of the transition of gastric parietal cells from the acid-secreting state to the resting state. Using purified gastric tubulovesicles and primary cultured rat parietal cells, we demonstrate for the first time that NHE3 prevents excessive tubulovesicular acidification and promotes membrane remodeling. These findings provide new insight into the homeostasis of parietal cells in the regulation of gastric acid secretion.

The kidney plays a central role in maintaining systemic homeostasis by regulating the excretion of metabolic compounds. In mammals, this is achieved through filtration followed by selective reabsorption, whereas insect Malpighian tubules (MTs) are thought to function primarily through active secretion. Although reabsorption of ions and water has been described in MTs, whether these tissues regulate metabolite balance through reabsorption remains unclear. Here, we uncover an amino acid (AA) reabsorption mechanism in Drosophila MTs. Quantification of AAs in excreta reveals that AA excretion changes rapidly and markedly in response to dietary protein levels, while systemic AA levels remain stable. Using ex vivo assays, we show that MTs actively transport AAs. We identify the AA transporter Slimfast (Slif), expressed in the lower tubule, as a key mediator required to maintain systemic levels of basic AAs. These findings reveal a previously unrecognised reabsorption-based strategy in insect renal tubules that functionally parallels mammalian kidney physiology, despite their distinct organisation. Our study demonstrates that AA excretion forms an additional layer to achieve metabolic homeostasis.

Voltage-gated proton channel Hv1/VSOP has long been regarded as a plasma membrane protein that modulates intracellular pH and membrane potential to support immune cell function. Here, we reveal an unexpected intracellular pool of Hv1 on endosomal membranes in microglia, where it orchestrates a reciprocal interplay between endosomal trafficking and the actin cytoskeleton. Combining endosome patch-clamp recordings with high-resolution imaging, we demonstrate that functional endosomal Hv1 forms tight and dynamic associations with F-actin. Genetic deletion of Hv1 markedly elongates actin filaments, a phenotype that appears to depend on intracellular rather than plasma membrane Hv1 activity. Heterologous expression of wild-type Hv1, but not a proton-non-conducting mutant, reduced the F-actin staining, indicating that the ion-conducting function is required for this regulation. Live-cell imaging reveals that Hv1-positive endosomes move in concert with F-actin networks and frequently engage with their terminal regions, suggesting that filament barbed ends are trapped at Hv1-positive endosomes. Proximity-labeling proteomics identifies the actin-capping protein CAPZ as a critical mediator of Hv1-dependent actin remodeling, and genetic ablation of CAPZ abolishes the actin phenotype in Hv1-deficient microglia. These findings uncover a previously unrecognized ion channel–cytoskeleton crosstalk that shapes endosomal function and microglial physiology, redefining the functional landscape of voltage-gated proton channels.

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While D-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal D-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a D-serine transporter previously uncharacterized in the kidney and discovered D-serine as a noncanonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal D-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

In humans, uric acid is an end-product of purine metabolism. Urate excretion from human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here we show that renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all renal tubular cells expressed the same set of urate transporters. Only 32% of renal tubular cells were related to both reabsorption and secretion, while the remaining renal tubular cells were related to either reabsorption or secretion, at 5% and 63% respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on cell-units. Our findings also suggest that three different tubular cell populations cooperate to regulate urate excretion from human kidney. Highlight/Key points: We identified three distinct cell populations within the human renal anatomy that predict putative cellular transport mode, and our findings indicate cellular inhomogeneity with distinct roles such as urate secretion and reabsorption. Our model of physiological urate handling demonstrates the excretion dynamics in human kidney in terms of single cell-units. Our cellular urate transport analyses suggest the reversibility of some urate transporters even in certain physiological conditions. The physiological function of SLC2A9 is not limited to urate reabsorption; it is also involved in urate secretion restriction. This methodology can be applied to investigations of transport mechanisms in general, regardless of epithelial cell types, species, and substrates.

Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson’s disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H⁺,K⁺-ATPase. The K⁺-dependent ATPase activity and the lysosomal K⁺-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase, thapsigargin, and K⁺-competitive inhibitors of gastric H⁺,K⁺-ATPase, such as vonoprazan and SCH28080. Interestingly, these H⁺,K⁺-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H⁺/K⁺-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes.

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b^0,+AT, which constitute system b^0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca²⁺ mediates higher-order assembly of system b^0,+. Ca²⁺ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b^0,+AT–rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca²⁺ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b^0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)–based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.