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

Authors

Jasmine N Tutol, Vishaka Pathiranage, Shelby M Phelps, Alice R Walker, Sheel C Dodani

Published in

bioRxiv : the preprint server for biology. Sep 17, 2026. Epub Sep 17, 2026.

Abstract

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.

PMID:
42779728
Bibliographic data and abstract were imported from PubMed on 16 Oct 2026.

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