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Fig. 7. Selective arylation of hyperreactive thiols by CPM disables the transmembrane redox sensor. Recording of channel activity starts from undefined transmembrane redox potential in the presence of 7 n-M Ca2+ (bar 1, both panels). In left panel [GSH]/[GSSG] = 3 mtf/1 mM (giving -180 mV redox potential) is symmetrically applied into both cis and trans (bar 2); subsequent addition of GSH 9.72 mM introduces —210 mV into cis (bar 3); extensive perfusion of both cis and trans is performed to remove transmembrane redox potential (bar 4) followed by reestablished with [GSH]/[GSSG] = 3 mM/1 mM to both cis and trans (bar 5). In right panel [GSH]/[GSSG] of 0.95 mM/0.10 mM, and 4.0 mM/1.79 mM are introduced into the cytoplasmic chamber for —180 mV in cis and trans, respectively (bar 2). Further reduction of cis to -220 mV is made by addition of [GSH]/[GSSG] to 4.55 m/W/0.1 mM (bar 3). After perfusion of both chambers to removed GSH and GSSG, CPM (20 nM) is introduced into cis for 2 min before being terminated by perfusion (bar 4, marked with an asterick). Reinstillation of [GSH]/[GSSG] is made with 0.95 mM/0.10 mM and 4.0 mM/1.79 mM in cis and trans, respectively (symmetrical — 180 mV; bar 5).

redox sensor.7 Figure 7 shows P0 graphs of two separate channels before and after labeling with CPM. Both channels respond strongly to symmetric -180 mV transmembrane redox potential and are negatively regulated by a —220/—180 mV cisltrans gradient [compare Fig. 7, bars 1-3 (left) and P0 graphs (right)]. After exposing the channels to 20 nM CPM for 2 min [Fig. 7, bar 4 (marked with an asterisk), CPM removed from the bath by extensive perfusion of the chamber with 25 volumes of buffer], neither channel responds to a -180/-180 mV symmetric cisltrans redox gradient (compare bars 4 and 5 in Fig. 7).

Acknowledgments

This work was supported by NIH Grants 2R01AR43140 and 1POAR17605.

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