Ryanodine Receptor Channel-Dependent Glutathione Transport in the Sarcoplasmic Reticulum of Skeletal Muscle Miklo ´s Csala,* Rosella Fulceri,† Jo ´zsef Mandl,* Angelo Benedetti,† ,1 and Ga ´bor Ba ´ nhegyi* *Department of Medical Chemistry, Pathobiochemistry and Molecular Biology, Semmelweis University, P.O. Box 260, H-1444, Budapest, Hungary; and †Dipartimento di Fisiopatologia e Medicina Sperimentale, Universita ` di Siena, 53100 Siena, Italy Received August 21, 2001 We found that glutathione transport across endo/ sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione in- flux could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 ago- nists. The effect of blockers was specific to glutathi- one, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarco- plasmic reticulum. © 2001 Academic Press Variations in intracellular Ca 2+ concentrations rep- resent one of the most important second messenger pathways in living organisms. A major mechanism for increasing cytosolic Ca 2+ is the release of Ca 2+ from internal stores (endoplasmic or sarcoplasmic reticu- lum, ER or SR) via the members of a superfamily of intracellular calcium-release channels including ryan- odine receptors (RyR) (1). Hypersensitive thiols of RyRs are subjects of oxidoreduction, which cause the activation or inhibition of Ca 2+ release (2–5). Generally speaking, thiol oxidation by reactive oxygen species, glutathione disulfide (GSSG), and other thiol reagents activate, while reducing agents, such as glutathione (GSH), dithiothreitol and mercaptoethanol, inhibit the channel. RyR type 1 (RyR1) from skeletal muscle can function as a transmembrane redox sensor (6). A large transmembrane redox potential inhibits, while dissipa- tion of this potential activates the channel. Therefore, it is logical to consider that GSH/GSSG transport across the SR membrane is involved in regulating the local redox potential gradient necessary for the redox regulation of RyR1. GSH and GSSG constitute the most important redox buffer of animal cells both in the cytosol and in or- ganelles. The transport of glutathione through endo- membranes is a less explored field of cell biology. Its importance is especially evident in the regulation of particular redox potentials found in each intracellular compartment. In a typical mammalian cell, the ratio of [GSH]/[GSSG] in the cytosol is 30 –100:1 resulting in a redox potential of about -230 mV. The lumen of ER is more oxidized (-180 mV) with a 1–3:1 ratio of [GSH]/ [GSSG] (7). The tightly controlled redox potential is essential, not only for the regulation of the RyR1 cal- cium channel in the SR of skeletal muscle, but also in the oxidative folding of secretory and membrane pro- teins in the ER lumen in liver and other secretory organs (8 –10). We have reported that GSH is transported through the membrane of hepatic ER at a relatively slow rate, while the membrane is practically impermeable toward GSSG (11). Recent data showed that both compounds could permeate the membrane of SR vesicles from skeletal muscle, although with different velocity (6). Here we report evidence for the involvement of RyR1 in GSH/GSSG transport across the SR membrane of skeletal muscle. We observed that the initial rate of GSH and GSSG transport is higher in terminal cyster- nae vesicles, which have a higher relative abundance of RyR1. As well, activators and inhibitors of the RyR1 calcium channel increase or decrease, respectively, the rate of transport. We suggest that RyR1 may behave as a glutathione transporter on its own, or alternatively Abbreviations used: RyR, ryanodine receptor; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; GSH, reduced glutathione; GSSG, glutathione disulfide; Mops, 4-morpholinepropanesulfonic acid. 1 To whom correspondence should be addressed at Dipartimento di Fisiopatologia e Medicina Sperimentale, Viale A. Moro n°1, 53100 Siena, Italy. Fax: +0577 234009. E-mail: benedetti@unisi.it. Biochemical and Biophysical Research Communications 287, 696 –700 (2001) doi:10.1006/bbrc.2001.5648, available online at http://www.idealibrary.com on 696 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.