A gain-of-function mutation in ATP6V0A4 drives primary distal renal tubular alkalosis with enhanced V-ATPase activity
Si-qi Peng, … , Xiao-liang Zhang, Bin Wang
Si-qi Peng, … , Xiao-liang Zhang, Bin Wang
Published April 29, 2025
Citation Information: J Clin Invest. 2025;135(13):e188807. https://doi.org/10.1172/JCI188807.
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Research Article Genetics Nephrology

A gain-of-function mutation in ATP6V0A4 drives primary distal renal tubular alkalosis with enhanced V-ATPase activity

Abstract

The ATP6V0A4 gene encodes the a4 subunit of vacuolar H+-ATPase (V-ATPase), which mediates hydrogen ion transport across the membrane. Previous studies have suggested that mutations in ATP6V0A4 consistently result in a loss of function, impairing the hydrogen ion transport efficacy of V-ATPase and leading to distal renal tubular acidosis and sensorineural hearing loss. Here, we identified a 32-year-old male patient and his father, both of whom harbored a heterozygous ATP6V0A4 p.V512L mutation and exhibited hypochloremic metabolic alkalosis, acidic urine, and hypokalemia. Through a series of protein structural analyses and functional experiments, the V512L mutation was confirmed as a gain-of-function mutation in the ATP6V0A4 gene. V512-a4 increased a4 subunit expression abundance by enhancing V512L-a4 stability and reducing its degradation, which in turn potentiated the capacity of V-ATPase to acidify the tubular lumen, leading to acidic urine and metabolic alkalosis. Through mutant V512L-a4 subunit structure-based virtual and experimental screening, we identified F351 (C25H26FN3O2S), a small-molecule inhibitor specifically targeting the V512L-a4 mutant. In conclusion, we identified a gain-of-function mutation in the ATP6V0A4 gene, broadening its phenotypic and mutational spectrum, and we provide valuable insights into potential therapeutic approaches for diseases associated with ATP6V0A4 mutations.

Authors

Si-qi Peng, Qian-qian Wu, Wan-yi Wang, Yi-Lin Zhang, Rui-ning Zhou, Jun Liao, Jin-xuan Wei, Yan Yang, Wen Shi, Jun-lan Yang, Xiao-xu Wang, Zhi-yuan Wei, Jia-xuan Sun, Lu Huang, Hong Fan, Hui Cai, Cheng-kun Wang, Xin-hua Li, Ting-song Li, Bi-cheng Liu, Xiao-liang Zhang, Bin Wang

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Figure 2

Molecular dynamics simulations and validation of patient kidney samples reveal V512L variant increases a4 protein stability and expression abundance.

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Molecular dynamics simulations and validation of patient kidney samples ...
(A) The prediction outcome of V512L-a4 stability was ΔΔG:0.268 kcal/mol (stabilizing). (B) Prediction of interatomic interactions of p.V512L. Residues 512 in the WT-a4 and V512L-a4 proteins are colored in light green and are shown as sticks. The respective chemical interactions are labeled as dotted lines and colored as follows: hydrogen bonds—(red), weak hydrogen bonds—(orange), hydrophobic contacts—(green), amide-amide contacts—(blue), and ionic interactions—(gold). Amino acid residues are also colored according to type, namely: nitrogen (blue), oxygen (red), and sulfur (yellow). In comparison with WT sites, increased interactions were observed to be added in mutant sites. (C) The WT-a4 has an average RMSD of 1.03 nm, and the V512L-a4 is 0.76 nm. (D) The WT-a4 has an average Rg of 4.500 nm, and the V512L-a4 is 4.455 nm. (E) For the residues between 500 and 530 near the V512L, the WT-a4 has an average RMSF of 0.473 nm, and the V512L-a4 is 0.456 nm. (F) Free energy landscapes for WT-a4 and V512L-a4. The free energy landscape uses a color gradient from blue (indicating high stability, folded states) to red (indicating low stability, unfolded states). IHC (G) and immunofluorescence (H) validated patient renal tubular tissues with increased ATP6V0A4 expression abundance compared with MCN, IgAN, and FSGS. Each group was always compared with the proband, which was considered as the reference group. Scale bar: 40 μm in IHC and immunofluorescence. Violin plots indicate median (red) and upper and lower quartile (blue). ***P < 0.0005, ****P < 0.0001 by 2-tailed unpaired t test. V512L, Val512Leu; RMSD, root mean square deviation; Rg, radius of gyration; RMSF, root mean square fluctuations; MCN, minimal change nephropathy; IgAN, IgA nephropathy; FSGS, focal segmental glomerulosclerosis.