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 ( K d = 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 β-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.