A Fluorescent-Based Method to Assess the Activity of the Human Sodium-Coupled Citrate Transporter

Abstract

Increasing evidence supports the human sodium-coupled citrate transporter (hNaCT) as a potential therapeutic target for metabolic syndrome and early infantile epileptic encephalopathy type 25 (EIEE25). While isotopic tracers remain the reference method for evaluating citrate transport, the need for specialized equipment and regulatory approval restricts their widespread use. Here, we report the development and validation of a robust, fluorescent-based assay to evaluate hNaCT-mediated citrate transport in live cells at both 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 a standard multiwell plate reader. A key strength of this approach is its ability to assess citrate transport in the same cells before and after experimental interventions. Accordingly, this approach enables the functional characterization of hNaCT, including its pharmacological inhibitors and genetic variants with altered activity. Overall, the method provides a reliable assessment of citrate transport and offers a versatile platform suitable for identifying novel lead compounds for the therapeutic modulation of hNaCT.

Increasing evidence supports the human sodium-coupled citrate transporter (hNaCT) as a potential therapeutic target for metabolic syndrome and early infantile epileptic encephalopathy type 25 (EIEE25). While isotopic tracers remain the reference method for evaluating citrate transport, the need for specialized equipment and regulatory approval restricts their widespread use. Here, we report the development and validation of a robust, fluorescent-based assay to evaluate hNaCT-mediated citrate transport in live cells at both 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 a standard multiwell plate reader. A key strength of this approach is its ability to assess citrate transport in the same cells before and after experimental interventions. Accordingly, this approach enables the functional characterization of hNaCT, including its pharmacological inhibitors and genetic variants with altered activity. Overall, the method provides a reliable assessment of citrate transport and offers a versatile platform suitable for identifying novel lead compounds for the therapeutic modulation of hNaCT.