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bioRxiv · 10.1101/2025.08.02.668312

A cell-based method to assess the activity of the human sodium-coupled citrate transporter

Carcamo-Lemus, n.·Bernier, A.·Oporto-Ortega, I.·San Martin, A.·Sandoval, P. Y.
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Abstract

Increasing evidence supports the human sodium-coupled citrate transporter (hNaCT) as a potential therapeutic target for metabolic syndrome, while genetic polymorphisms in hNaCT have been associated with neurological disorder. Isotopic tracers remain the primary method for evaluating citrate transport, which are labor-intensive, require dedicated equipment, and demand highly skilled personnel. Herein, we develop and validate a robust, live-cell-compatible fluorescent method to evaluate citrate transport activity mediated by hNaCT at single-cell and high-throughput levels. This method utilizes a baculoviral vector to modify HEK293 cells to co-express a genetically encoded citrate sensor (Citron1) and the hNaCT. This cell-based platform enabled real-time monitoring of citrate transport using fluorescent microscopy and standard multiwell plate reader. The present method enables the functional characterization of hNaCT gain- and loss-of-function mutation, as well as the evaluation of pharmacological inhibitors targeting citrate transport. These results demonstrate that the cell-based method reliably assesses citrate transport at a single-cell resolution and is compatible with high-throughput screening, to identify novel lead compounds for the therapeutic modulation of hNaCT.

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Carcamo-Lemus, n., Bernier, A., Oporto-Ortega, I., San Martin, A., Sandoval, P. Y.. 2025-08-03. A cell-based method to assess the activity of the human sodium-coupled citrate transporter. https://doi.org/10.1101/2025.08.02.668312

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