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 iodidequenching assays, leaving real-time imaging of chloride transport a largely unexplored frontier. To address this gap, here, 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 response (13.9-fold) while boosting affinity (Kd = 47.4 mM) and bound-state brightness (13.6). Molecular dynamics simulations provide atomic-level insights for these enhancements, revealing that 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 to monitor chloride transport under basal and pharmacologically inhibited conditions in the U-2 OS cell model. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/710669v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1635d96org.highwire.dtl.DTLVardef@954ea5org.highwire.dtl.DTLVardef@5e6b5borg.highwire.dtl.DTLVardef@da29b5_HPS_FORMAT_FIGEXP M_FIG C_FIG