Cis

trans trans

Fig. 4. Schematic representation of the bilayer chambers with incorporated single channel in planar phospholipid membrane.

glutathione.7 The hyperreactive cysteine moieties constitute the essential component of the redox sensor, which conveys information about localized changes in redox potential produced by physiologic (e.g., glutathione) and pathophysiologic (e.g., quinones) channel modulators of the Ca2+ release process.10'12'13

Measurement of Transmembrane Redox Sensor of Channels Reconstituted in Bilipid Layer Membrane

The bilayer lipid membrane (BLM) preparation affords precise control of the redox state on both the cytoplasmic and lumenal faces of the reconstituted channel. Adjustment of the GSH : GSSG ratio to form various redox potentials on either side of the membrane has turned out to be a powerful approach for detecting the RyR redox sensor. Our modified method for preparing junctional SR (i.e., including GSH/GSSH in the homogenization solution to buffer redox potential to ~0.22 V) protects against overoxidation of critical sulfhydryls, and thus provides a higher percentage of "intact" channels that monitor and functionally follow transmembrane redox fluctuation.

incorporation of Ryanodine Receptor 1 in Planar Lipid Bilayer

Reconstitution of RyR 1 into BLM and recording of the channel activity are performed at room temperature (24°). The typical BLM setup is composed of a chamber and a removable cup and can be purchased commercially (Warner, Hamden, CT) (Fig. 4). The bilayer cup possesses a small circular hole (200-300 /¿m) and can be snugly inserted into one of the machined holes of the chamber, thereby forming two compartments whose only electrical connection is the hole in the

13 I. N. Pessah, C. Beltzner, S. W. Burchiel, G. Srihar, T. Penning, and W. Feng (2001). Mol. Pharmacol. 59, 507 (2001).

cup. Typically Cs+ in the form of CsCl is used as charge carrier, and the current is measured as a holding potential is applied to the cis (cytoplasmic) chamber of the reconstituted channel whereas the trans (lumenal) chamber is the virtual ground. In the standard commercially available BLM setup (e.g., Warner), the cis and trans chambers each have a 0.7-ml capacity. Specifically, two standard stock solutions are prepared and stored at 4° for up to 4 weeks:

cis solution: 500 mM CsCl, 100 pM CaCl2, 20 mM HEPES; adjusted to pH 7.4

trans solution: 50 mM CsCl, 100 pM CaCl2, 20 mM HEPES; adjusted to pH 7.4

Once the cis and trans solutions are in place (10:1 Cs+ gradient), the BLM is made with 5 :2 phosphatidylethanolamine: phosphatidylcholine (50 mg/ml in decane; Northern Lipids, Vancouver, BC, Canada). It is formed by adsorbing a small amount of the lipid solution on either a small sable brush trimmed to two or three bristles or a Pasteur pipette that has been fired to shape a small ball at its end. The brush or pipette is gently brought across the hole within the bilayer cup. Lipid solutions are made fresh daily and used once. To reconstitute a channel, the cis side is held to a positive potential relative to the trans (ground) side, which is favorable for JSR vesicle fusion with the BLM. On fusion of JSR with BLM, the cis-to-trans Cs+ gradient is reversed to 1:10, by purfusing the cis chamber with 15 volumes of 50 mM CsCl, 20 mM HEPES, pH 7.4 (no added CaCl2), and the trans chamber with 500 mM CsCl, 20 mM HEPES, pH 7.4. A negative holding potential is applied to the cis side and single-channel activity is measured with a bilayer amplifier [e.g., Dagan (Minneapolis, MN) 3910 Expander], The data are filtered at 1 kHz before being acquired at 10 kHz by a Digidata 1200 (Axon Instruments, Union City, CA). The data are analyzed by pCLAMP 6 (Axon Instruments) without additional filtering.

Preparation of Reduced and Oxidized Glutathione Stock Solutions

GSH is dissolved in degassed HEPES (20 mM) buffer, and the solution is adjusted to pH 7.0. Aliquots (~0.2 ml) are portioned into vials briefly gassed with argon and sealed. Vials can be stored at —20° for up to 60 days without measurable oxidation of GSH. Each vial is used once. GSSG solutions are made and stored in a similar manner except that degassing and argon are omitted. When used as transmembrane glutathione redox buffer in bilayer lipid membrane (BLM) experiments, GSH and GSSG are sampled from both sides of the BLM chamber at the end of channel recordings to verify that the initial redox potential has not changed during the course of the experiments. Redox buffers have been found to be stable for at least 1 hr. GSH and GSSG contents are determined according to the method of Senft et al.,14 using the fluorescence indicator o-phthalaldehyde (OPA).

Experimental Design to Obtain Redox Potential

The glutathione redox potential is calculated by the Nernst equation from the GSH: GSSG ratios and the total glutathione concentration according to

[GS Jz where £h is the redox potential referred to the normal hydrogen electrode, V; Eq is the standard potential of glutathione, —0.24 V; R is the gas constant, 8.31 J/deg • mol; T is the absolute temperature (K); n is the number of electrons transferred, n = 2 for SH-SS exchanges; F is the Faraday constant, 96,406 J/V; and [GSSG] and [GSH] are the molar concentrations of oxidized and reduced glutathione, respectively.

At the typical temperature for bilayer measurements (22°),

[OS"] = ([GSSG]/10(£h+0'24)/29'28)1/2 [GSSG] = [OS"]2 x io^+a24>/29-28

Instillation of Glutathione Redox Gradient across Bilipid Layer Membrane

Single-channel studies have shown that glutathione used as an individual reducing (GSH) or oxidizing (GSSG) agent can inhibit or stimulate RyR gating activity, respectively.15 However, this approach does not address the physiological importance of glutathione in the regulation of RyR channel function. In mammalian cells, the redox buffer system is composed of the glutathione couple, GSH and GSSG. Furthermore, the microsomal membrane within which RyR and IP3R reside is normally subject to a large transmembrane redox potential difference with a high ratio of GSH to GSSG in the cytosol, >30:1, and a much lower and stabilized ratio of 3 :1 to 1:1 maintained within lumen.

These physiological conditions can be easily mimicked in the BLM preparation. A precise combination of GSH and GSSG can be instilled on each side of the BLM to precisely set the transmembrane glutathione redox potential. Furthermore, the redox potential in trans can be maintained at a physiologic —180 mV whereas the cis potential can be varied by perfusion of solutions possessing defined ratios of GSH to GSSG. Using this approach the typical reconstituted RyRl channel in

14 A. P. Senft, T. P. Dalton, and H. G. Shertzer, Anal. Biochem. 280, 80 (2000).

15 A. C. Zable, T. G. Favero, and J. J. Abramson, / Biol. Chem. 272, 7069 (1997).

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