Glycoprotein Triadin Involves Redox Cycling ofFunctionally. Important .... diluted 100-fold (50 pg/ml) in solution A consisting of 100 mM KCl, 20 m~ MOPS, pH ...
THEJOURNAL OF BIOUXICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 52, Issue of December 30, pp. 33028-33034, 1994 Printed in U.S.A.
Molecular Interaction between Ryanodine Receptorand Glycoprotein Triadin Involves Redox Cycling ofFunctionally Important Hyperreactive Sulfhydryls* (Received for publication, August 12, 1994, and in revised form, October 14, 1994)
Guohua Liu and IsaacN. PessahS From the Department of Molecular Biosciences, Division of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, California 95616
Thefluorogenicmaleimide 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM) has been shown to selectively form Michaeladducts with hyperreactive sulfhydryls onthe skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994)Mol. Pharmacol. 45,189-200). The present report demonstrates a functionally important interaction between RyRl and triadin which involves,in part,redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyRland triadin only in the presence of Ca2+channel inhibitors ( M e , neomycin, ruthenium red, or anti-triadin antibody). Treatment ofSR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics >lO-fold,2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex(HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyRl and triadin by Western blot and immunoprecipitation analyses. Highaffinity [$H]ryanodine-bindingsites are immunoprecipitated by either anti-RyR1 or anti-triadinantibody dose dependently. 1,4-Naphthoquinone (540 pmoypg protein) selectively oxidizeshyperreactive sulfhydryls on RyRl and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by j3-mercaptoethanol or dithiothreitol into its component RyRl and triadin protomers. The results provide direct evidence for the existence of a functionally important complex between RyRl and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1-triadin complexcan signal information across the SR membrane.
Ryanodine-sensitive Ca2+ channels( i e . ryanodine receptors; RyR1)’ of skeletal muscle sarcoplasmic reticulum (SR) arelocalized at T-tubuleISR junctions where they constitute the junctional “foot”protein, a homooligomer of four 565-kDa(based on cDNA) protomers (1-3). The purified RyRl protein reconstituted into bilayerlipid membranes maintains many of the pharmacological sensitivities characteristic of the native calcium release channel found in SR membranes. However, “purified channels exhibit gating kinetics which are quantitatively different from native channels studied SR in membranes subconductance and havea higher tendency to exhibit multiple states (4-6). Anomalies in Ca2+channel function are evenmore accentuated whenexpressed from RyRl cDNA transfected into COS-1 and Sf9 cells (7-8). Functionally important interactions between RyRl and distinct triadic proteins may underlie differences in the functions of native, purified, and heterologously expressed channel preparations. There is emerging evidence that the Ca2+release channel oligomer is structurally associated with distinct junctional proteins which may serve t o initiate, regulate, and terminate the Ca2+release process during excitation-contraction coupling. Triadic proteins which have been shown to be directly or indirectly associated with, and influence, the function of RyRl include: 1)the al-subunitof the L-type voltage-dependent Ca2+ channel of the T-tubule membrane (i.e. the dihydropyridine receptor) (9, 10); 2) the immunophilin FKBPl2 (FK-506 binding protein-12 kDa) (11-15); 3) the principal Ca2+binding protein of the SR lumen, calsequestrin (16, 17); and 4)the glycoprotein triadin (18-23). Of these proteins, triadin has been proposed to provide the bridging structure which links the dihydropyridine receptor and RyRl (18-19, 23), although the exact functional role of triadin in excitation-contraction coupling is unclear. Recently Liu et al. (24) demonstrated theexistence of a discrete class(cl pmol/pg of SR protein) of highly reactive (hyperreactive)thiolgroups which areprimarily localized on the RyRl protomer and triadin using the fluorogenic maleimide 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM). Utilizing CPM at concentrations significantly lower than the free thiol pool found in membrane preparations enriched in terminal cisternae (i.e. 0.02-1.0 pmol CPWpg protein), the rate of formation of fluorescent CPM-thio adducts was found to be highly dependent on the presence of physiological modulators of RyR1. Under conditions which promote
* This work was supported in part by National Institutes of Health The abbreviations usedare: RyR1, skeletal isoform of the ryanodine Grant ES05002 and National Institute of Environmental Health Sciences Center Grant ES05707. The costs of publication of this article receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l-propane were defrayed in part by the payment of page charges. This article must sulfonate; CPM, 7-diethylamino-3-(4’-maleimidylphenyl~-4-methylcoutherefore be hereby marked “aduertisement” in accordance with 18 marin; MOPS, 3-(N-morpholino)propanesulfonicacid; HMWC, high molecular weight complex; NQ, 1,4-naphthoquinone; RR, ruthenium red; U.S.C. Section 1734 solely toindicate this fact. PAGE, polyacrylamide gel electrophoresis; $ To whom correspondence should be addressed: Dept. of Molecular SR, sarcoplasmic reticulum; Biosciences, University of California, Davis, CA 95616. Tel.: 916-752- Dm, dithiothreitol; PVDF, polyvinylidine difluoride; PBS, phosphatebuffered saline. 6696; Fax: 916-752-4698.
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Hyperreactive Sulfhydryls Regulate .lYiadin RyRl Complex
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channel activation (e.g. nanomolar ryanodine, micromolar (2%p-mercaptoethanolor 2 mM DTT) in the sample buffer andboiled for Ca2+),addition of 0.2 pmol of CPWpg of protein resulted inslow 3 min prior to electrophoresis. The fluorescent protein bands on PAGE adduct formation with the abundant Ca2+-ATPase. Conditions gels werevisualized at 360 nm excitation using a transilluminator and the fluorescence image was photographed through a 450-nm cutoff filwhich promote channel closure (millimolar M e , millimolar ter. The fluorescence intensity of protein hands was digitizedby a video Ca2+,or micromolar ryanodine) resulted in extremely rapid analysis system (Jandel Scientific, Corte Madera, CAI and integrated labeling of a single class of hyperreactive sulfhydryls localized by computer within the linear range of protein density. Electroelution of CPM-labeledSRProtein-FollowingSDS-PAGE, primarily on RyRl and triadin (24). Formation of thio-adducts between CPM and the hyperreactive sulfhydryls on RyRl and fluorescent bands on the gels were visualizedat 360 nmexcitation using triadin was shown to markedly alter normal channel gating, a transilluminator to identify CPM-labeled bands. When the bands of interest were not labeled by CPM,1or 2 laneswere cut from the gel and revealing the essential role of hyperreactive sulfhydryl chem- stained with Coomassie Blue to serve as a reference to permit the bands istry in the channel gating process (24). These initial results of interest to be located in the unstained lanes. The bands of interest with CPM suggested a functional role for a RyRl.triadin com- were carefully cut from the gel with a razor blade, and chopped into plex. Differencesin labeling kinetics with membrane-permeant small pieces. Electroelution was performed at -10 &glass tube at CPM may reflect titration of a very small number of thiols (
