TMEM175 Knockout Hela Cell Line

The TMEM175 Knockout Hela Cell Line is a CRISPR/Cas9-edited knockout cell line featuring targeted disruption of the TMEM175 gene. This loss-of-function model prevents functional TMEM175 expression, providing a stable genetic background for studying lysosomal pH regulation and autophagy. Engineered via CRISPR/Cas9 technology, the line eliminates interference from the endogenous protein, enabling precise dissection of TMEM175-dependent pathways.

Derived from the HPV18-positive HeLa cervical adenocarcinoma cell line, this knockout retains the robust growth and high transfection efficiency characteristic of its host. HeLa cells are widely used in gene-editing studies due to their well-characterized genetic landscape. This epithelial origin offers a relevant model for investigating lysosomal dynamics and mTOR signaling in a cancer context, while supporting reproducible phenotypic analyses of lysosomal dysfunction pathways.

TMEM175 encodes an endolysosomal K+ channel that regulates lysosomal membrane potential and pH. By balancing V-ATPase-mediated proton pumping, TMEM175 supports optimal activity of hydrolases like cathepsin B and facilitates the lysosomal recruitment of mTORC1 via the Ragulator?CRag GTPase complex. Disruption results in lysosomal alkalinization, impaired autophagic flux, and diminished mTORC1 signaling. The transcription factor TFEB both promotes TMEM175 expression and, upon lysosomal stress, undergoes altered nuclear translocation, creating a regulatory feedback loop that integrates lysosomal ion balance with cellular catabolic programs.

In the HeLa context, TMEM175 knockout models lysosomal dysfunction relevant to Parkinson disease, where TMEM175 variants are risk factors. Loss of channel function recapitulates reduced lysosomal clearance and mTORC1 dysregulation observed in neurodegeneration. Although HeLa is non-neuronal, it conserves the autophagy-lysosome-mTOR axis, permitting detailed mechanistic dissection and small-molecule screening for lysosomal enhancers targeting the TMEM175 pathway.

This knockout line is suited for autophagy assays (LC3-II/p62 western blotting, bafilomycin A1 flux), lysosomal pH measurement (LysoSensor), cathepsin B activity tests, and mTORC1 signaling readouts (phospho-S6K1 immunoblotting, LAMP1-mTOR colocalization). Applications include Parkinson disease modeling, mTOR signaling studies, and drug screening for lysosomal modulators. Its stable genetic perturbation supports high-content and complementation experiments. For details, contact Ascent Research.