Non-thiol reagents regulate ryanodine receptor function by redox interactions that modify reactive thiols.

The Ca(2+) release channel (CRC) from sarcoplasmic reticulum (SR) is rich in thiol groups, and their oxidation/- reduction by thiol reagents activates/inhibits the CRC. Most channel regulators are not thiol reagents, and the mechanism of their action is illusive. Here the authors show that many channel activators act as electron acceptors, while many channel inhibitors act as electron donors in free radical reactions. The channel activator, caffeine, and the CRC inhibitor, tetracaine, are shown to interact competitively, which suggests that there exists a common site(s) on the CRC, that integrates the donor/acceptor effects of ligands. Moreover, channel activators shift the redox potential of reactive thiols on the ryanodine receptor (RyR) to more negative values and decrease the number of reactive thiols, while channel inhibitors shift the redox potential to more positive values and increase the number of reactive thiols. These observations suggest that the non-thiol channel modulators shift the thiol-disulfide balance within CRC by transiently exchanging electrons with the Ca(2+) release protein.

Reactions with dye free radicals reveal weak redox properties of drugs.

The calcium release channel (CRC) of the skeletal sarcoplasmic reticulum is rich in thiol groups and is strongly regulated by covalent modification of these thiols. Oxidizing reagents activate the release channel, whereas reducing reagents inhibit the channel. However, most CRC regulators are not thiol reagents. Here, we propose that reversible redox interactions are involved in regulation of the CRC by nonthiol reagents. This hypothesis was tested with several CRC regulators. The local anesthetics tetracaine, procaine and QX-314, which block the CRC, behaved as electron donors in reactions with dye free radicals. In contrast, ryanodine, caffeine, doxorubicin and daunorubicin, compounds known to activate the release channel, all accepted electrons from dye anion radicals. Moreover, release of Ca2+ from SR initiated by doxorubicin (acceptor) was antagonized by local anesthetics (donors). Based on the redox characterization of CRC modulators, we propose a functional model in which channel inhibitors and activators act as weak electron donors and acceptors, respectively, and shift the thiol-disulfide balance within the release protein. The consequence of this model is that, in spite of the chemical diversity of CRC modulators, a common mechanism of channel regulation involves the transient exchange of electrons between the activator/inhibitor and the CRC.

Functional distinctions between the mitochondrial ATP-dependent K+ channel (mitoKATP) and its inward rectifier subunit (mitoKIR).

The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK(ATP)) is a highly selective conductor of K(+) ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoK(ATP) is inhibited with high affinity by ATP (K((1/2)) = 20-30 microM). We have suggested that holo-mitoK(ATP) is a heteromultimer consisting of an inwardly rectifying K(+) channel (mitoKIR) and a sulfonylurea receptor (Grover, G. J., and Garlid, K. D. (2000) J. Mol. Cell. Cardiol. 32, 677-695). Here, we show that a 55-kDa protein isolated by ethanol extraction and reconstituted in bilayer lipid membranes and liposomes is the mitoKIR. This protein, which lacks the sulfonylurea receptor subunit, is inhibited with low affinity by ATP, with K(1/2) approximately 550 microM. ATP inhibition of both mitoKIR and holo-mitoK(ATP) is reversed by UDP (K((1/2))1/2 = 10-15 microM). Holo-mitoK(ATP) is and diazoxide, and the opened by cromakalim flux through the open channel is inhibited by glibenclamide and 5-hydroxydecanoate. None of these agents has any effect upon mitoKIR. We have identified two compounds that act specifically on mitoKIR. p-diethylaminoethylbenzoate reverses inhibition of mitoKIR by ATP and ADP at micromolar concentrations and also opens mitoK(ATP) in isolated mitochondria. Tetraphenylphosphonium inhibits K(+) flux through both mitoKIR and mitoK(ATP) with the same apparent affinity. These findings support the hypothesis that the 55-kDa mitoKIR is the channel component of mitoK(ATP).