100 msec


100 msec


B 100 msec

(1) 40 |iM Ca2* ]?5pA Po = 0.320 cis . trans ma^Mm^*^^** o i -1 -1 I!iWPWÜPilfW1wnMNVf«' PWPÜ


Fig. 6. Responses of the high-P0 gating mode channel to transmembrane redox signaling. In (A), after instillation of transmembrane redox potential, the trans side is held constant at — 180 mV potential by 4.0 mM GSH and 1.792 mM GSSG. On the cis side, -220 mV is formed by 4.0 mM GSH and 0.077 mM GSSG (trace 2), and -180 mV is formed by 4.0 mM GSH and 1.792 mM GSSG. In (B), -220 mV on the cis side is formed by 4.0 mM GSH and 0.077 mM GSSG, and -180 mV on the trans side is formed by 4.0 mM GSH and 1.792 mM GSSG. The voltage holding potential is held at -30 and —40 mV for (A) and (B), respectively.

probable that NQ initially enhances channel activity through a redox-sensing mechanism whereas the subsequent effects are the result of protein arylation.12 The unique response of channels exhibiting the high-P0 gating mode to NQ can be tested by perfusing both sides of the BLM with buffer lacking GSH and GSSG (100 ixM Ca2+, 1 mM Mg2+ in cis, Pa = 0.037; trace 3, Fig. 6B). Addition of 5 [iM NQ reveals that high-/3,, channels that fail to respond to glutathione transmembrane redox potential also fail to exhibit an initial activation phase with

NQ (P0 = 0.032; trace 4, Fig. 6B). These channels, however, are eventually inhibited irreversibly (P0 < 0.001; trace 5, Fig. 6B). Taken together these results suggest that redox-sensing cysteine moieties within the RyRl complex are easily overoxidized (probably to disulfides) with an attendant loss of the redox sensor.

Association of Hyperreactive Sulfhydryls with Ryanodine Receptor Transmembrane Redox Sensor

As shown above, conditions promoting the closed state of the channel permit the rapid formation of thioether adducts between CPM and RyR thiols. Whether these hyperreactive thiols are an essential component of the redox sensor can be tested. A single channel is reconstitued in BLM and tested for redox sensing as described above. Nanomolar CPM is then added to the cis chamber under low-P0 conditions to promote CPM arylation of hyperreactive thiols. Although CPM does not appear to alter channel P0 and gating kinetics, it does disable the transmembrane

cis trans l-l-l

Open probability

Open probability cis trans l-l-l

0 0

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