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Fig. 5. RyRl responses to transmembrane redox potential regardless of the glutathione concentration applied in the buffer. Free Ca2+ concentrations present in eis buffer are 7, 50, and 7 ßM for channels in (A), (B), and (C), respectively. Holding potentials of —30, -40, and -40 mV were applied to channels shown in (A), (B), and (C), respectively. Current fluctuation is downward. R.P. (mV), Redox potential (in millivolts).

BLM exhibits a tight response to changes in transmembrane redox potential independent of absolute concentrations of GSH and GSSG. It is important to note that control of reconstituted RyRl channels has been observed to be lost with high (non-physiologic) concentrations of glutathione (>5 mM GSH and GSSG). Figure 5 demonstrates the modulation of RyRl channel activity by experimentally setting transmembrane redox potential. In Fig. 5A, the current through the RyRl channel is recorded in the presence of 7 \xM free Ca2+ at a holding potential of —40 mV. One second of representative current traces from a total of 2-5 min of recording is displayed. The channel in the absence of defined redox potential (without glutathione redox buffer on either side of the channel) displays a low open probability, P0 (trace 1). GSH (2.5 mM) and GSSG (0.7 mM) introduced into the cis solution give a redox potential of -180 mV (calculated according to the Nernst equation), and has a negligible influence on channel activity (trace 2). Subsequent introduction of the same GSH: GSSG ratio (approximate redox potential of — 180 mV) into the trans chamber significantly increases channel P0 (trace 3). Perfusion of the cis side with a reduced redox potential of -220 mV (4.0 mM GSH and 77 ¡¿M GSSG) results in a dramatic decrease in P0 (trace 4). Figure 5B and C show that the RyR channel is activated on instillation of a symmetric —180 mV transmembrane redox potential, and is independent of the individual GSH and GSSG concentration used in the cis and trans chambers to obtain —180 mV (compare trace 1 with trace 2 in Fig. 5B and C).

Measurement of Redox Sensing of Ryanodine Receptor Channel in High Open Probability Gating Mode

Studies have revealed that RyRl channels in a high-/\, gating mode reflect an overoxidized state of the protein. Even though glutathione is included in the initial homogenization buffer to avoid this potential problem, critical free sulfhydryls are likely to be oxidized in the room air during subsequent handling of JSR preparations, and frequently channels exhibiting a high-P0 gating mode are observed. With many but not all channels, instillation of transmembrane redox potential resets the redox sensor of high-/3,, type channels. Figure 6 shows examples of two channels, one that recovers redox sensing and another that does not. Figure 6A shows a typical high-/3,, channel in the presence of 50 ¡iM cis Ca2+ but with undefined transmembrane redox potential (P0 of 0.637; trace 1). Instillation of a relatively reduced redox potential gradient cis to trans (—220 to —180 mV, respectively) immediately results in a Iow-Pq gating mode (P„ of 0.031; trace 2, Fig. 6A). A subsequent change in the cis/trans transmembrane redox potential to — 180/— 180 mV clearly demonstrated recovery of the transmembrane redox sensor (P0 increases to 0.125; trace 3, Fig. 6A).

By contrast, Fig. 6B shows another channel exhibiting the high-P0 gating mode (P0 = 0.320 with 40 fxM cis Ca2+; trace 1). Introducing a cis/trans redox potential of -220 mV/—180 mV does not affect channel gating activity (P0 = 0.32; trace 2, Fig. 6B). The lack of responsiveness to transmembrane redox potential exhibited by this type of channel may reflect the fact that the critical (hyperreactive) cysteines that are an essential component of the redox sensor may be damaged by overoxidation to disulfides. Addition of a low concentration of redox-active 1,4-naphthoquinone (NQ) has been shown to produce time-dependent biphasic actions on RyR gating activities. NQ activates RyR channels rapidly (a reversible effect) followed in time by complete inactivation (an irreversible effect). It is

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