o-Phthalaldehyde Activates the Ca 2+ Release Mechanism from Skeletal Muscle Sarcoplasmic Reticulum Jonathan J. Abramson,* ,1 Shawn P. Mullen,* Steffen Koehler,* David Mansoor,† Peter Anderson,† Carl C. Wamser,‡ Trudy J. Swan,§ and Terence G. Favero† *Department of Physics and ‡Department of Chemistry, Portland State University, Portland, Oregon 97207; §Biology Department, McKay High School, Salem, Oregon; and †Department of Biology, University of Portland, Portland, Oregon 97203 Received January 31, 2001, and in revised form April 11, 2001; published online June 13, 2001 o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approxi- mately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca 2 -ATPase activity at low micromolar concen- trations and induces Ca 2 release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concen- tration dependence. At low OPA concentrations (< 100 M), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time- dependent sequential activation and inhibition of re- ceptor binding is observed. Activation is character- ized by a Ca 2 -independent increase in maximal recep- tor occupancy. Data are presented to support a model in which Ca 2 channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine resi- dues. OPA complexation with endogenous lysine resi- due(s) is critical for receptor activation. © 2001 Academic Press Key Words: ryanodine receptor; Ca 2 release chan- nel; excitation-contraction coupling. Skeletal Muscle sarcoplasmic reticulum (SR) 2 is re- sponsible for controlling the cytosolic Ca 2+ concentra- tion and hence the contractile state of muscle. By rap- idly releasing its stores of Ca 2+ through the ryanodine receptor (RyR1), it is directly responsible for causing muscle contraction. By actively accumulating Ca 2+ via Ca 2+ , Mg 2+ -ATPase, the SR lowers the cytosolic Ca 2+ concentration to submicromolar levels which leads to muscle relaxation. The Ca 2+ release mechanism of SR has been shown to be sensitive to the cellular redox environment (1–3). Addition of heavy metals (4, 5) and sulfhydryl oxidizing reagents such as Cu 2+ /mercaptan, phthalocyanine dyes, reactive disulfides, anthraquinones, and porphy- rins directly interact with the Ca 2+ release protein from the SR (6 –10). Sulfhydryl oxidation to a disulfide causes the Ca 2+ permeability of the SR and the open probability of the reconstituted release channel to in- crease. Reduction of the disulfides formed causes the membrane to once again become impermeable to Ca 2+ (6, 7, 9, 10) and the release channel to close. Sulfhy- dryl-induced Ca 2+ release is also modulated by all the known effectors of Ca 2+ release. Using nanomolar con- centrations of the fluorogenic coumaryl maleimide, CPM, Lui et al. (11) have identified a class of “hyper- reactive” thiols that are specifically labeled in the closed state of the channel. Functional changes associ- ated with N-ethyl maleimide and diamide reacting 1 To whom correspondence should be addressed at Physics Depart- ment, Portland State University, P.O. Box 751, Portland, Oregon 97207-0751. Fax: (503) 725-3888. E-mail: abramsonj@pdx.edu. 2 Abbreviations used: OPA, o-phthalaldehyde; SR, sarcoplasmic reticulum; RyR1, ryanodine receptor type 1; Hepes, N-(2-hydroxy ethyl)piperazine-N'-(2-ethanesulfonic acid); Pipes, piperazine-N, N'- bis (2-ethanesulfonic acid); APIII, antipyrylazo III; PE, phosphati- dylethanolamine; PS, phosphatidylserine; DMSO, dimethylsulfox- ide; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N, N, N' , N' - tetraacetic acid; ME, -mercaptoethanol; TCEP, tris-(2-carboxy ethyl)phosphine hydrochloride; CPM, 7-diethylamino-3-(4'-male- imidylphenyl)-4-methylcoumarin; BLM, bilayer lipid membrane; EC 50 , excitatory concentration—50%; IC 50 , inhibitory concentra- tion—50%; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, FITC, fluorescein 5-isothiocyanate; NBD-Cl, 7-chloro-4-nitrobenz-2-oxa-1,3,-diazole; DNFB, 2,4-dinitrofluoro benzene. 0003-9861/01 $35.00 235 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 391, No. 2, July 15, pp. 235–244, 2001 doi:10.1006/abbi.2001.2403, available online at http://www.idealibrary.com on