Cryo-Electron Microscopy Study of the human Citrate Transporter NaCT

Abstract

NaCT, a membrane transporter within the SLC13 family, plays a critical role in cellular metabolism by mediating sodium-dependent citrate transport. In this study, I conducted a comprehensive analysis of human NaCT (hNaCT) through structural, functional, and inhibitory assays. The purified hNaCT protein was reconstituted into nanodiscs for structural studies, revealing its inward-facing conformation at a resolution of 2.75 Å via Cryo-electron microscopy (Cryo-EM). Functional assays highlighted the critical influence of sodium and pH gradients on citrate transport, with maximum activity observed at pH 6.0 and an external sodium concentration of 200 mM. nano-Differential Scanning Fluorimetry (nanoDSF) experiments demonstrated that SLC13A5-IN-1 provided the highest thermal stabilization for hNaCT at pH 7.5. However, transport assays identified PF-06649298 as the most potent inhibitor of hNaCT activity, followed by PF- 06761281, BI01383298, and SLC13A5-IN-1. The discrepancy between thermal stability and inhibitory efficacy may arise from differences in solubility and binding mechanisms. These findings offer novel insights into the structure and function of hNaCT, providing a foundation for the development of targeted therapeutics for metabolic disorders. Further studies are needed to refine inhibitor binding models and evaluate their clinical potential.