St Lsr

SERCA Triadin

■ Calsequestrin

Fig. 2. SDS-PAGE pattern of light and junctional SR fractions isolated by sucrose density gradient centrifugation, using the method of A. Saito, S. Seiler, A. Chu, and S. Fleischer, J. Cell Biol. 99, 875 (1984).

However, we have found that when [CPM] is limiting (0.02-1 pmol/jLtg SR) the kinetics of thioether adduct formation differ significantly with protein composition of the SR (light SR vs. junctional SR; see below) and the composition of the assay medium (presence of RyR activators or inhibitors).

Isolation of Junctional Sarcoplasmic Reticulum Membranes: Importance of Redox-Bujfered Solutions

The most commonly used experimental preparation consists of membrane vesicle fractions isolated from either fast twitch skeletal muscle of New Zealand White rabbit or cardiac muscle from the right ventricle obtained from a variety of species (rat, mouse, rabbit, sheep, etc.). For example, skeletal membranes derived primarily from junctional regions of SR (JSR) and enriched in ryanodine receptor type 1 (RyRl) and its associated proteins are routinely isolated by sucrose density gradient centrifugation within the 38-45% interface as described in detail by Saito et al.4 and Chu et al.5 A light SR (LSR) membrane fraction enriched in SR/ER Ca2+ ATPase (SERCA) pump but lacking RyRl complex can also be isolated from the 32-34% sucrose interface from the same five-step gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis reveals a clear distinction between the protein composition of the two fractions (Fig. 2). Junctional SR enriched in RyR2 complex and longitudinal SR deficient in RyR2 complex can be isolated from heart muscle in a similar manner.6

4 A. Saito, S. Seiler, A. Chu, and S. Fleischer, J. Cell Biol. 99, 875 (1984).

5 A. Chu, M. C. Dixon, A. Saito, S. Seiler, and S. Fleischer, Methods Enzymol. 157, 36 (1988).

6 M. Inui. S. Wang, A. Saito, and S. Fleischer, Methods Enzymol. 157, 100 (1988).

We have included reduced and oxidized glutathione (GSH and GSSG, respectively) in the initial skeletal muscle homogenization solution to buffer its redox potential.7 GSH (3.5 mM) and GSSG (59 ¡iM) are added to homogenization solution consisting of 0.3 M sucrose, 5 mM imidazole-HCl (pH 7.4), 100 ¡iM phenyl-methylsulfonyl fluoride (PMSF), and leupeptin (10 /¿g/ml) to give a redox potential (£h) of —0.22 V (see below for calculation of redox potential). This redox potential mimicks the typical cytoplasmic redox potential in vivo and is likely to prevent overoxidation of the RyR complex during the early stages of membrane isolation. The redox buffer is included only in the initial homogenization and first 1 l,000g pellet wash; it is omitted in subsequent steps. The inclusion of a redox buffer in the initial homogenization solution yields JSR preparations that possess a high fraction of reconstituted channels exhibiting a low open probability (low-P0) gating mode (>40%) that can be tightly regulated by transmembrane redox potential. By comparison, conventional preparations lacking redox buffer possess a high fraction of reconstituted channels exhibiting a high-P0 gating mode whose behavior appears to stem from overoxidization of protein thiols in the isolation protocol.7-10 This observation is consistent with a report that a physiological concentration of GSH partially protects the RyRl complex from oxidation in room air. Under ambient oxygen tension (PO2 ~ 150 mmHg) RyRl loses about six free thiols per subunit compared with when it is under physiological Po2 10 mmHg, normally found in muscle).10 Therefore the high-P0 gating mode more frequently observed with conventional JSR preparations may actually represent channels in an overoxidized state.

Kinetic Measurement of Hyperreactive Sulfhydryls of Junctional Sarcoplasmic Reticulum, Using 7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin

CPM is dissolved in dry dimethyl sulfoxide (DMSO) at a final stock concentration of 100 /iM, and 50-/il aliquots are divided into opaque plastic microcentrifuge tubes and stored for up to 1 year at —20°. When needed, an aliquot of stock is diluted serially 100-fold with DMSO just before use. All stocks are protected from light by wrapping the container with aluminum foil. The kinetics of CPM-thioether adduct formation with JSR or LSR are determined with a fluo-rimeter equipped with a 3-ml cuvette holder, which can provide precise control of temperature and stirring of the contents [e.g., SLM (Urbana, IL) model 8000 or Hitachi (Tokyo, Japan) model F-2000]. Excitation and emission are set at 397 and 465 nm (width of slit, 4 nm), respectively. It is advantageous to interface the

7 w. Feng, G. Liu, P. D. Allen, and I. N. Pessah, J. Biol. Chem. 275, 35902 (2000).

8 J. J. Marengo, C. Hidalgo, and R. Bull, Biophys. J. 74, 1263 (1998).

9T. Murayama, T. Oba, E. Katayama, H. Oyamada, K. Oguchi, M. Kobayashi, K. Otsuka, and

10 J. P. Eu, J. Sun, L. Xu, J. S. Stamler, and G. Meissner, Cell 102, 499 (2000).

instrument with a computer possessing data acquisition and analysis software. In our laboratory, the Hitachi F-2000 captures fluorescence data at 1 Hz. The data files are exported for nonlinear regression analysis by Origin 6.0 software (Microcal Software, Northampton, MA).

To quantify the kinetics of CPM-thioether adduct formation, SR membrane vesicles (50 fig of protein per milliliter) are incubated in a solution consisting of 100 mM KC1 and 20 mM 3-(A^-morpholino)propanesulfonic acid (MOPS), pH 7.0, at 37°. Modulators of RyR channel activity are added to the assay as small aliquots from a >100x stock, using a Hamilton syringe, and permitted to equilibrate for

I min. CPM is quickly added by Hamilton syringe through the opening in the cover to the spectrofluorometer directly into the center of the stirring sample to give a final concentration of 1-50 nM. Under these conditions (0.02-1 pmol of CPM per microgram of SR protein), the concentration of free SR sulfhydryls greatly exceeds that of CPM, and permits direct analysis of highly reactive cysteine residues under the conditions that enhance or inhibit RyR channel activity.2 '011 Figure 3 shows that the modulation of the rate of thioether adduct formation with JSR is highly dependent on whether a channel inhibitor (2 mM Mg2+) or a channel activator (50 ¡jlM Ca2+) is included in the assay buffer. By contrast, LSR deficient in RyR complex is insensitive to buffer conditions and exhibits only slow kinetics for forming CPM-thioether adducts regardless of whether channel-activating or -inhibiting conditions are present (Fig. 3, bottom). SDS-PAGE analysis of CPM-labeled skeletal JSR quenched within 1 min of initiation of the reaction reveals that RyRl and triadin form CPM-thioether adducts primarily during the rapid phase (when channel conditions favor the closed state). When channel activation is favored, the slow phase of adduct formation is primarily on the abundant SERCA pump (Fig. 3, top, inset).

The time course of the increase in fluorescence intensity (Ft) obtained under conditions promoting channel closure (millimolar Mg2+ or Ca2+ buffered to <100 nM by EGTA2) or channel activation (in the presence of micromolar Ca2+ or redox-active quinone12) is fitted with single or multiexponentials, respectively, leading to the corresponding time constants (k) from which apparent half-times (tm) are calculated (Fig. 3, top). The rate constant (k) was considered to be proportional to the number of free sulfhydryl groups available for CPM conjugation (i.e., k = fcm[SH]t).2 A unique feature of the hyperreactive thiol moieties is that Ca2+ and ryanodine, known to activate and inhibit channel activity depending on concentration, allosterically influence the rate of adduct formation in a biphasic manner.2 Studies reveal the existence of a transmembrane redox sensor within the RyRl channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and lumenal

II G. Liu and I. N. Pessah, J. Biol. Chem. 269, 33028 (1994).

12 W. Feng, G. Liu, R. Xia, J. J. Abramson, and I. N. Pessah, Mol. Pharmacol. 55, 821 (1999).

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