Pathiranage, V. | Search bioRxiv

bioRxiv2026

An All-Optical Approach to Probe Chloride Transport with a Bright ChlorON

Chloride transport across cellular membranes is fundamental to physiology. Yet, this dynamic process remains difficult to capture with existing methods that rely on electrophysiology or indirect iodide-quenching assays, leaving real-time imaging of chloride transport a largely unexplored frontier. To address this gap, we upgrade our first-generation fluorescent protein indicator ChlorON-1 into ChlorON-1-PRO through targeted mutagenesis of an evolutionarily conserved gatepost residue. A single mutation (C139N) preserves the turn-on sensing mechanism (13.9-fold response) while boosting affinity (Kd = 47.4 mM) and bound-state brightness (13.6). Molecular dynamics simulations provide atomic-level insights for these enhancements, supporting a model in which the mutation globally rigidifies the {beta}-barrel and locally prearranges the binding pocket while stabilizing the chromophore. Finally, we showcase the utility of ChlorON-1-PRO for real-time monitoring of endogenous chloride transport under basal and pharmacologically modulated conditions in the U-2 OS cell model.

biochemistry↗

bioRxiv2024

Genetically Encoded Red Fluorescent Indicators for Imaging Intracellular and Extracellular Potassium Ions

Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in E. coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 3.55 mM and 14.81 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K+ dynamics in various cell types and cellular environments.

biochemistry↗