Redox Regulation of Ca2 Release from Sarcoplasmic Reticulum

Several studies on the redox regulation of the SR Ca2+-release channel complex, consisting of the RyR, the FK506 binding protein and other associated proteins and molecules such as junctin, triadin and calmodulin, have been conducted on skeletal and cardiac muscle cells. Feng et al. have provided direct evidence that ryanodine channel activity follows transmembrane redox potential (28). The molecular basis of 02-responsiveness in these cells was identified to involve 6-8 of 50 RyR thiols whose redox state is dynamically controlled, as described by Eu et al. (23). Oxygen exposure of native SR leads to activation of the RyR-coupled release channel due to the oxidation of 6 RyR thiols per subunit and enhanced ryanodine binding to RyR across the spectrum of physiological concentrations. From their studies on SR vesicles, Abramson and Salama (1, 2) proposed a model for redox regulation of the channel that involves three different sulfhydryl groups which exist in close proximity and react through thiol oxidation and thiol-disulfide interchange to open or close the channel. Biochemical evidence suggests that the SR channels in cardiac myocytes can also be regulated by the redox potential of the cells.

Boraso and Williams demonstrated that H202 increased the open probability of the Ca2+-release channel in sheep and the channel activity was suppressed by DTT (Fig. 3) (11). Although they reported the involvement of H202 in the process of oxidative modification of the RyR, it is not clear if H202 itself reacted with RyR or that some product of H202, probably the hydroxyl radical is the true reactive agent. The effect of H202 and DTT on [Ca2+]cyt in cardiomyocytes is consistent with our observations on the tension of the ductus arteriosus (DA) (60).

Figure 3. Modification of the gating of the cardiac SR Ca2+-release channel by H202 and DTT. A: Representative current fluctuation in control (a) and after addition of 12 mM EGTA (A). 5 mM H202, applied to the cytosolic side of membrane, reopened the channel (c). B: Single channel recordings before (a) and after addition of DTT (b). Under these conditions H202 was not able to reopen channels (c). Application of ATP (d) and caffeine (e) increased the open probability of channels. Cardiac SR Ca2+-release channels were activated by 10 |iM Ca2+. Holding potential for single-channel recording was 0 mV. Dashed lines, closed-channel level; arrows, open-channel level (From Ref. 11).

Figure 3. Modification of the gating of the cardiac SR Ca2+-release channel by H202 and DTT. A: Representative current fluctuation in control (a) and after addition of 12 mM EGTA (A). 5 mM H202, applied to the cytosolic side of membrane, reopened the channel (c). B: Single channel recordings before (a) and after addition of DTT (b). Under these conditions H202 was not able to reopen channels (c). Application of ATP (d) and caffeine (e) increased the open probability of channels. Cardiac SR Ca2+-release channels were activated by 10 |iM Ca2+. Holding potential for single-channel recording was 0 mV. Dashed lines, closed-channel level; arrows, open-channel level (From Ref. 11).

H202 constricted and DTT dilated DA rings consistently. The opposite effects of H202 and DTT on DA rings would indicate a potential mechanism for normoxia-induced vasoconstriction and hypoxia-induced vasodilatation in DA. Suzuki et al. showed that H202 enhanced Ca2+ release from SR (65). The augmentation of Ca2+ release requires the thiol reductant DTT or glutathione (GSH), suggesting that thiol-disulfide exchange reactions regulate Ca2+ release from the SR by converting the disulfide structure to another thiol through reduction (by GSH or DTT) and oxidation (H202) reactions. This is consistent with the proposal by Abramson and Salama in skeletal muscle that thiol-disulfide interchange reactions within the Ca2+-release channel molecule convert the open/closed states of the channel (1, 2). Consistent with this observation is the observation that thiol reductants DTT and GSH suppress SR Ca2+-release channel activity (11, 83). The results shown that redox modification of RyR channels affect Ca2+ signaling in cardiac and skeletal muscle. In both cases, oxidation produces a significant increase in Ca2+ release. Reversible activation of cardiac RyR channels by oxidation could be relevant in the heart, especially when there is an increase in free radical production such as in ischemia/reperfusion situations. Sustained or uncontrolled oxidation of RyR channels may elicit an imbalance in Ca2+ homeostasis, which could trigger apoptosis.

Wilson et al. presented a possible new mechanism for acute hypoxia-sensing in PASMCs involving Ca2+ release from ryanodine-sensitive SR stores. These observations suggest that acute hypoxia increases a-NADH levels, which then increase the net amount of cyclic ADP-ribose (cADPR) synthetized from P-NAD+ by cADP-ribosyl cyclase, and simultaneously inhibit cADP degradation. The increased level of cADPR promotes Ca2+ release from RyR and elicits vasoconstriction (76).

The influence of ROS on Ca2+ release through IP3R-coupled channels has been examined in smooth muscle cells. Kaplin et al. (35) investigated redox modulation of Ca2+release in purified IP3 receptors reconstituted in lipid vesicles. Reduced nicotinamide adenine dinucleotide (NADH) increased IP3-mediated Ca2+ flux. [Ca2+]cyt was markedly and specifically increased by direct intracellular injection of NADH, suggesting that direct regulation of IP3 by NADH could be responsible for elevated [Ca2+]cyt occurring in the earliest phase of hypoxia.

These findings suggest that in the mechanisms that are necessary for sensing and responding acutely to hypoxia, both intracellular and extracellular are important. However, the nature of the redox-related protein modification is in question and the molecular details are not known. It seems likely that sensitive thiol-groups within the channel complexes are an essential component of a transmembrane redox sensor and may be involved in mediating changes in Ca2+-signaling during changes in Po2.

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