Oxidation and Reduction of Pig Skeletal Muscle Ryanodine Receptors

13 Pages·1999·0.19 MB·English

3010 BiophysicalJournal Volume77 December1999 3010–3022
Oxidation and Reduction of Pig Skeletal Muscle Ryanodine Receptors
Claudia S. Haarmann,* Rainer H. A. Fink,# and Angela F. Dulhunty*
*MuscleResearchGroup,JohnCurtinSchoolofMedicalResearch,Canberra,ACT2601,Australia,and#SecondInstituteofPhysiology,
UniversityofHeidelberg,HeidelbergD69120,Germany
ABSTRACT Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from
normal pigs and RyR (Arg615 to Cys615) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents
MH
4,49-dithiodipyridine (4,49-DTDP) and 5,59-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT).
NormalandRyR channelsrespondedsimilarlytoallreagents.DTNB(1mM),eithercytoplasmic(cis)orluminal(trans),or
MH
1 mM 4,49-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,49-DTDP (cis), but not DTNB,
inhibited channels after .5 min. Activation and inhibition were relieved by DTT (1–10 mM). DTT (10 mM, cytoplasmic or
luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained
with1mMDTNBand10mMDTTpresentonthesameoroppositesidesofthebilayer.Wesuggestthat1)4,49-DTDPand
DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian
skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either
cytoplasmorlumen,resideinthetransmembraneregion;4)somecardiacsulfhydrylsareunavailableinskeletalRyRs;and
5)Cys615inRyR isfunctionallyunimportantinredoxcycling.
MH
INTRODUCTION
Contraction of striated muscle depends on Ca21 release or b-mercaptoethanol (BME). The domain of the RyR on
from the sarcoplasmic reticulum (SR), through ryanodine which oxidants act is not clear. Both lipid-soluble and
receptor (RyR) calcium release channels. Oxidants are im- water-solublereagentscanpartitionintothemembrane,toa
portantligandsinthephysiologicalactivityofRyRsandact greater or lesser extent, depending on their pK values and
a
by oxidation of free sulfhydryl (SH) groups, because their couldreactwith-SHgroupsoneitherthesidetowhichthey
effectsarepreventedbysulfhydrylreducingagents(Boraso areaddedorinthetransmembranepartsoftheRyRprotein.
and Williams, 1994; Favero et al., 1995; Abramson et al., Itisunlikelythatreagentswouldactrapidlyontheopposite
1995).OxidantsactivateandcaninhibitRyRs(Holmberget side of the bilayer, because reagent crossing the bilayer
al., 1991; Holmberg and Williams, 1992; Boraso and Wil- would be diluted in the large volume of the opposite solu-
liams,1994;Faveroetal.,1995;Stoyanovskyetal.,1997). tionandwouldtakealongtimetoreachactivatingconcen-
Oxidation-induced activation proceeds under in vivo con- trations. Water-soluble oxidants appear to react with SH
ditions,inthepresenceofglutathione(GSH)(Koshitaetal., groups only in the cytoplasmic or transmembrane domains
1993), and is enhanced during ischemia or reperfusion, in
of the mammalian RyRs. Methanethiosulfonate derivatives
which oxygen free radicals increase and the ratio of GSH:
block skeletal muscle RyRs only from the cis side (Quinn
GSSG falls (Sies et al., 1972; Curello et al., 1985).
andEhrlich,1997).Someresidues(- SHand-SH)respon-
a i
Reagents that react specifically with free SH groups are
sibleforactivationorinhibitionofcardiacRyRsareacces-
usedasmodelcompoundstolookattheeffectsofoxidation
sible to thimerosal from either side of the bilayer and are
and the importance of cysteine residues in RyR channel
located in the transmembrane domain, while other activat-
function. Reactive disulfides 4,49- or 2,29-dithiodipyridine
ing(- *SH)residuesareconfinedtothecytoplasmicdomain
(4,49-DTDPor2,29-DTDP)activateandthenblockcardiac a
(Eager and Dulhunty, 1999). There have been no similar
RyRs when applied to the cytoplasmic (cis) side of the
reports of the long-term effects of thiol-specific oxidizing
channels (Eager et al., 1997; Eager and Dulhunty, 1998).
agents added to the luminal or cytoplasmic sides of mam-
Skeletal RyRs have also been reported to be activated, but
malian skeletal RyR channels.
not inhibited, by cis 4,49- or 2,29-DTDP and by GSSH
Thepresentstudyexaminestheeffectsonsingleskeletal
(Nagura et al., 1988; Marengo et al., 1998) and to be
RyRs of long exposure to 2–10 mM concentrations of the
inhibitedbythereducingagentsGSH,dithiothreitol(DTT),
thiol-specific 4,49-DTDP or 5,59-dithiobis-(2Nitrobenzoic
acid) (DTNB) or DTT. The experiments tested the hypoth-
esis that, as in cardiac RyRs (Eager et al., 1999), three
Receivedforpublication10May1999andinfinalform19August1999. classes of -SH (- SH and -SH, within the transmembrane
a i
AddressreprintrequeststoDr.A.F.Dulhunty,DivisionofBiochemistry domain, or - *SH in the cytoplasmic domain) are available
a
andMolecularBiolgy,JohnCurtinSchoolofMedicalResearch,Australian
in skeletal RyRs for oxidation by specific sulfhydryl re-
NationalUniversity,P.O.Box334,Canberra,ACT2601,Australia.Tel.:
agents. It was possible that the same thiols could be oxi-
61-2-6249-4491; Fax: [phone]; E-mail: angela.dulhunty@anu.
edu.au. dized in cardiac and skeletal RyRs, because 71 of .80
© 1999bytheBiophysicalSociety cysteine residues are conserved between the two isoforms
0006-3495/99/12/3010/13 $2.00 (Otsu et al., 1990). The experiments tested an additional
Haarmannetal. OxidationandReductionofSkeletalRyRs 3011
hypothesisthattheArg615-to-Cys615substitutioninRyR throughcottongauzeandcentrifugedat10,0003gfor30min.Thepellet
MH
frompigssusceptibletomalignanthyperthermia(MH)pro- (P2)wascollected,andthesupernatantwascentrifugedagainat35,0003
g and the pellet (P3) collected. Pellets P2 and P3 were resuspended in
vides an additional -SH group for oxidation. MH is an
(mM) 5 Tris-2-(N-morpholino)ethanesulfonic acid (Tris-MES), 300 su-
inherited skeletal muscle disorder, characterized by in-
crose,100KCl,2DTT(pH6.8).Aliquotsofthesuspensionswerefrozen
creasedCa21releasefromSR(Ohtaetal.,1989;Carrieret inliquidnitrogenandstoredat270°C.Allbufferscontainedtheprotease
al., 1991; Mickelson and Louis, 1996) and reduced inhibi- inhibitors phenylmethylsulfonyl fluoride (0.7 mM), leupeptin (1 mg/ml),
tionofRyRsbyCa21(Filletal.,1991;Shomeretal.,1993) pepstatinA(1mM),andbenzamidine(1mM).
or Mg21 (Laver et al., 1997).
Novel findings are 1) that the water-soluble DTNB (in
Lipid bilayer techniques
either cis or trans solutions) and the lipid-soluble 4,49-
DTDP (in the cis solution) activate skeletal RyRs in a Thelipidbilayerandsingle-channelrecordingtechniquearedescribedby
similarway;2)4,49-DTDPaddedtothecissolution,butnot Ahern et al. (1994) and Laver et al. (1995). Bilayers were formed from
DTNB(cisortrans),inhibitsskeletalRyRsafter.5min;3) phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine
(5:3:2w/w)(AvantiPolarLipids,Alabaster,AL)acrossanaperturewitha
in the absence of an oxidizing reagent, 10 mM DTT acti-
diameter of 200–250 mm in the wall of a 1.0-ml Delrin cup (Cadillac
vatesskeletalRyRsfromthecisortranssolution;4)“con- Plastics,Australia).TCvesicles(finalconcentration10mg/ml)wereadded
trol-like”channelactivityismaintainedwhen1mMDTNB tothecischamberandstirreduntilvesicleincorporationwasobserved.The
and10mMDTTarepresenttogetheroneitherthesameor cytoplasmic side of channels incorporated into the bilayer faced the cis
oppositesidesofthebilayer;and5)effectsofoxidationand solution.Thebilayerpotentialwascontrolledandsingle-channelactivity
wasrecordedwithanAxopatch200Aamplifier(AxonInstruments,Foster
reduction are the same in normal RyRs and RyR . Our
MH City,CA).Forexperimentalpurposes,thecischamberwasheldatground
conclusions are that 1) - SH is located in the skeletal RyR
a andthevoltageofthetranschamberwascontrolled.Bilayerpotentialis
transmembrane domain, as in cardiac RyRs; 2) - *SH, in expressedintheconventionalwayasV 2V (i.e.,V 2V ).
a cis trans cytoplasm lumen
cardiac RyRs, is not available for oxidation in skeletal Bilayerswereformedandvesiclesincorporatedintothebilayer,using
RyRs; 3) a modified thiol in the transmembrane domain cisandtranssolutionscontaining(mM)230Csmethanesulfonate(CsMS),
20 CsCl, 0.1 CaCl, and 10 N-tris[hyroxymethyl]methyl-2-aminoethane-
(- S-R, where R is either a protein S if the modified thiol 2
ab sulfonic acid (TES) (pH 7.4 adjusted with CsOH). The cis solution also
isadisulfide,oranNifthemodifiedgroupisnitrosylated;
contained500mMmannitoltoaidSRvesiclefusionandRyRincorpora-
Xu et al., 1998) normally suppresses skeletal RyR activity; tionintothebilayer.
and 4) the Arg615-to-Cys615 substitution in MH does not
provide an additional SH group for oxidation by thiol-
specific reagents. Recording and analysis of single-channel data
Channelactivitywasrecordedat240mV.Ingeneralactivitywasrecorded
fora2-mincontrolperiodbeforeand1minafter0.5-svoltagepulsesto
MATERIALS AND METHODS
140mV.Drugswerethenaddedtoeitherthecisortranschamberwitha
Biological material and caffeine-halothane ;10-sstirringperiod,andthenactivitywasrecordedforseveralminutes.
Voltagepulsesto140mVwereoccasionallyappliedtothebilayerafter
contracture test for MH susceptibility
drug addition to determine whether channels could be activated by the
change in bilayer potential. Because RyR channel activity can increase
The methods for genetic testing, muscle dissection, caffeine-halothane
immediately after a voltage pulse (Zahradnikova and Meszaros, 1998;
contracturetesting,preparationofSRvesicles,andsingle-channelrecord-
LaverandLamb,1998),thefirst30saftereachvoltagepulsewasexcluded
inghavebeendescribedpreviously(Otsuetal.,1992;Owenetal.,1997;
fromanalysis.
Laveretal.,1997).Muscleandbloodsampleswereobtainedfromthree
Channelactivitywasfilteredat1kHz(10-polelow-passBessel,23dB)
homozygousnormalpigs(oneBelgiumLandraceandtwoLandrace)and
anddigitizedat2kHz.Analysisofsingle-channelrecords(usingChannel
three homozygous MH pigs (two Belgium Landrace and one Landrace)
aged;4months.EachanimalwasgeneticallytestedforanormalorMH 2,writtenbyP.W.GageandM.Smith)yieldedchannelopenprobability
RyRallele(containingeitherArg615orCis615).TheSRpreparationswere (Po),frequencyofevents(Fo),opentimes,closedtimes,andmeanopenor
closedtimes(T orT),aswellasmeancurrent(I9).Theopendiscriminator
fromthesameanimalsasthoseusedinLaveretal.(1997).Thedescription o c
wassetat;25%ofthemaximumcurrent,andthecloseddiscriminatorat
of anesthetic techniques, muscle dissection, and halothane/caffeine con-
50%oftheopendiscriminator,sothatopeningstobothsubconductance
tracturetestsaregivenbyLaveretal.(1997).Allfiberbundlesfromthe
and maximum conductance levels were included in the analysis. Single-
threehomozygousnormalanimalsfailedtorespondtohalothaneor2mM
channelparametersweremeasuredduringthe30s,showingmaximumI9
caffeine,whileallfiberbundlesfromthethreehomozygousMHanimals
during the control period and then the period of maximum I9 after the
developedtensioninresponsetobothdrugs.
additionofdrugs.Dwell-timedistributionswereplottedasthefrequencyof
openings in logged bins (Sigworth and Sine, 1987) to display the large
Isolation of SR vesicles range of open times seen in control recordings and after the addition of
oxidizingreagents.Thefitofamultipleexponentialfunctiontothedata
The preparation of crude SR vesicles was based on the methods of wasassessedusingaleast-squaresfit.
Meissner(1984)andMaetal.(1995).Thefreshlydissectedbackandleg
muscle was washed in cold phosphate-buffered saline containing 2 mM
EGTA(pH7.0),trimmedoffatandconnectivetissue,cubed,andeither Statistics
frozeninliquidN andstoredat270°Corfreshlyprocessed.Thefreshor
2
thawedmusclecubesweresuspendedin(mM)5Trismaleate,100NaCl, Averagedataaregivenasmean6SEM.Thesignificanceofthedifference
2EDTA,0.1EGTA(pH6.8)(5ml/goftissue).Themusclewashomog- betweencontrolandtestvalueswasevaluatedwithaStudent’st-test,either
enizedinaWaringblenderinfour15-shigh-speedbursts.Thehomogenate one or two sided and for independent or paired data, as appropriate.
wascentrifugedat26003gfor30min,andthesupernatantwsfiltered Differenceswereconsideredtobesignificantwhenp#0.05.
3012 BiophysicalJournal Volume77 December1999
RESULTS
Effects of DTNB or 4,4*-DTDP on normal RyRs
ActivationbyDTNB
RyRs from homozygous normal pig muscle were activated
by addition of the hydrophilic DTNB (1 mM) to either the
cis or trans chamber. Control RyR activity was character-
izedbybriefopeningstothemaximumconductanceandto
lowerconductancelevels(Figs.1,AandB,and2,AandB).
LongeropeningsappearedwithDTNB(Figs.1Aand2B).
Channel activity increased with a delay of ;1 min after
DTNB was added to the cis chamber in 13 of 14 channels,
withadelayof;3minaftertransDTNBwasaddedto10
of 11 RyRs. Average mean current (I9) increased from a
controlvalueof21.4360.12pAto23.9160.19pA(n5
10)orfrom22.7360.38pAto25.8160.64pA(n56).
When the average ratio of mean current before and after
addition of the drugs for individual channels was deter-
mined, there was an approximately fivefold increase with
cis DTNB and an approximately threefold increase with
transDTNB(Table1).Notethatthedelaywasassessedby
FIGURE 2 InhibitionofnormalRyRsby1mMcis4,49-DTDP,butnot
by1mMtransDTNB.(A)AbilayercontainingoneRyRchannelunder
control conditions (I), activation 3 min after the addition of 1 mM cis
4,49-DTDPdissolvedinethanol(II),inhibition10minaftertheadditionof
4,49-DTDP(III),andrecoveryfrominhibitionafterafinaladditionof2
mMDTT(IV).(B)AbilayercontainingtwonormalRyRchannelswith
onlyonechannelactiveundercontrolconditions(I),increasedactivityin
one channel 3 min after the addition of 1 mM trans DTNB (II), two
channelsactiveaftera10-minexposuretotransDTNB(III),andreturnto
controlactivityinonechannelafterafinaladditionof10mMDTT(IV).
Thezerocurrentlevelisindicatedbysolidlines,andthemaximumopen
conductanceisshownbydottedlines.
eyefromchannelrecords.ThenumbersofchannelsforI9in
Table 1 are fewer than the number for the delay becauseI9
wasmeasuredusingChannel2analysisoflow-noiserecord-
ings only.
Channelactivitywith1mMcisDTNBwasnotalteredby
subsequent addition of 2 mM cis DTT (Results below) but
returnedtowardcontrolwhen10mMDTTwasaddedtothe
FIGURE 1 Activation of normal RyRs by 1 mM DTNB or 1 mM cis side of nine of nine RyRs activated by cis DTNB or to
4,49-DTDPaddedtothecischamberandreversalofactivationbyDTT.
the trans side of three of three RyRs activated by trans
Channelactivityinthisandsubsequentfigureswasrecordedat240mV
withsymmetrical250mMCs1and100mMCa21.(A)AsingleRyRunder DTNB(Figs.1and2).Thisfallinactivitywaspresumably
controlconditions(I),duringactivationby1mMcisDTNB(II)andthen duetoreductionof-SHgroupsoxidizedbyDTNB,because
afteradditionof10mMDTTtothecischamber(III).(B)AsingleRyR DTT (10 mM cis or trans) added without DTNB activated
under control conditions (I), during activation by 1 mM cis 4,49-DTDP, RyRs (Results below). The similar magnitude and time
addedfroma10mMstockdissolvedincissolution(II),andthenafterthe
course of effects ofcis or trans DTNB support the hypoth-
additionof2mMDTTtothecischamber(III).Thezerocurrentlevelis
esis that DTNB oxidizes - SH, located in the transmem-
indicatedbysolidlines,andthemaximumopenconductanceisshownby a
brokenlines. brane domain.
Haarmannetal. OxidationandReductionofSkeletalRyRs 3013
TABLE 1 Averageeffectofdrugsonmeancurrent,I*,innormalRyRs(normal)andRyR (MH)
MH
Normal MH
I9 /I9 (n) Delay[s] (n) I9 /I9 (n) Delay[s] (n)
drug control drug control
DTNB(cis) 5.2861.47** (10) 68613 (13) 3.9160.92** (9) 198638## (14)
DTNB(trans) 2.8760.73** (6) 207650 (10) 4.7061.42** (9) 141625 (16)
DTDP(ethanol) 6.5663.34** (12) 138648 (15) 4.2261.53* (7) 214635 (8)
Ethanol(1%) 2.6561.03* (5) 168621 (8) 1.7960.51 (6) 150613 (5)
DTDP(HO) 3.4161.64* (8) 100615 (18) 3.0260.87 (13) 156627 (13)
2
RelativechangesinI9(I9 /I9 )areshown.DTNBand4,49-DTDP(DTDP)wereaddedtoafinalconcentrationof1mM.Dataaregivenasmean6
drug control
1SEM.
I9 issignificantlydifferentfromI9 with:*p,0.05.**p,0.01.
drug control
I9 /I9 forRyR issignificantlydifferentfromI9 /I9 fornormalRyRswith:##p,0.01.
drug control MH drug control
DTDP(ethanol),DTDPdissolvedinethanol.
DTDP(HO),DTDPdissolvedincissolution.
2
Activationby4,49-DTDP at 240 mV, with 100 mM Ca21 and symmetrical 250 mM
CsMS(Fig.3).Single-channelconductancewas45269pS
4,49-DTDP(1mMcisaddedinethanol,1%v/v)causedan
(n 5 4) for normal RyRs and 470 6 11 pS (n 5 5) for
approximately sevenfold increase in I9, which began ;2
RyR ,andcontrolI9forRyR was1.7660.13pA(n5
min after its addition to 15 of 15 channels (Table 1). In MH MH
39), compared with ;2.1 pA for 16 normal RyRs (Results
contrast to sheep cardiac (Eager et al., 1997) or rabbit
above). In addition, both types of channel were 1) locked
skeletal (Ahern et al., 1997a) RyRs, 1% ethanol (alone)
into a submaximum conductance state with 10–15 mM cis
increased I9 after ;3 min in eight of eight RyRs (Table 1).
ryanodine(RyR,n511;RyR ,n512),2)blockedby5
Therefore 4,49-DTDP was dissolved in cis solution at 10 MH
mMandaddedtothecischambertoafinalconcentrationof
;1mM.Inthissituation,I9increasedin18of21channels,
after a delay of ;1.5 min (Table 1). Activity returned
towardcontrolafter2mMcisDTTadditiontoeachoffour
single4,49-DTDP-activatedRyRs(Figs.1Band2A).Thus
4,49-DTDP also activated RyRs via oxidation of -SH
groups, which could also be - SH in the transmembrane
a
domain.
Inhibitionby4,49-DTDP
Cis 4,49-DTDP (1 mM) abolished activity in seven of 10
RyRs, after 334 6 52 s (4,49-DTDP plus ethanol, Fig. 2
AIII), or in five of nine channels after 456 6 82 s (4,49-
DTDPaddedincissolution).Inactivatedchannelswerenot
reactivatedbyfourpulsesto140mV(0.03Hz).Infrequent
activityremainedinsevenof19channels,for26mininone
case. RyRs were not inactivated after 5–30 min with 1%
ethanol alone (n 5 9). Cis DTT (2 mM) restored activity
after 129 6 62 s in three of five RyRs inactivated by
4,49-DTDPplusethanol(Fig.2AIV)orafter2406165sin
threeoffiveRyRsinactivatedby4,49-DTDPalone.Recov-
ery with DTT shows that inactivation is due to -SH oxida-
tionandthat-SHispresentinskeletalRyRsandaccessible
i
toDTT.Incontrastto4,49-DTDP,1mMDTNB(eithercis
or trans) did not inactivate RyRs after 5–20 min (Fig. 2 B,
FIGURE 3 RyR is activated by 1 mM cis DTNB and 1 mM cis
II and III). If activity fell, it increased again after pulses to 4,49-DTDP.(A)AMsHingleRyR undercontrolconditions(I)andduring
140 mV. activationby1mMcisDTNBM(HII),andrecoveryaftertheadditionof10
mM DTT to the cis chamber (III). (B) A single RyR under control
MH
conditions(I),duringactivationby1mMcis4,49-DTDP,andaddedfrom
Comparison of RyR and RyR a10mMstockdissolvedincissolution(II),andrecoveryaftertheaddition
MH
of2mMDTTtothecischamber(III).Thezerocurrentlevelisindicated
The characteristics of normal RyRs and RyR (from ho-
MH bysolidlines,andthemaximumopenconductanceisshownbythebroken
mozygous MH pigs) were similar under control conditions lines.
3014 BiophysicalJournal Volume77 December1999
mMcisrutheniumred(RyR,n53;RyR ,n52),and3) channelsrecovered50626safter2mMDTTwasadded.
MH
activated by 5 mM ATP (RyR, n 5 4; RyR , n 5 5). No RyR channels were inhibited during 3.5–32-min ex-
MH MH
posure to DTNB (cis or trans, n 5 13) or 4–30-min expo-
sure to 1% cis ethanol (n 5 5). Thus inhibition of RyR
Effects of reactive disulfides on RyRs from
channels via oxidation of -SH was not altered by the MH
MH pigs i
mutation.
ActivationbyDTNBor4,49-DTDP There were some curious exceptions to the general ob-
servations reported above. One normal and two RyR
Activityincreasedfourfoldin14of14RyR channels;3 MH
MH channels were inactivated 2–7 min after 4,49-DTDP was
minaftertheadditionof1mMcisDTNBorapproximately
added,withoutinitialactivation,andinhibitionwasrelieved
fourfold in 16 of 17 channels ;2 min after trans DTNB
40–90saftercisDTTwasadded.Thisconfirmedindepen-
(Fig. 3, Table 1). Activation of RyR by cis DTNB was
MH dent channel activation and inhibition and supported sepa-
significantly slower than activation of normal RyRs (p ,
rate - SH and -SH residues (see also Eager et al., 1998).
0.01). There were no other differences in activation by a i
Activity in three RyR and two normal RyRs fell after
DTNB between normal RyRs and RyR . 4,49-DTDP MH
MH initial activation by cis 4,49-DTDP, and activity in two
added to RyR , with ethanol or alone (in cis solution),
MH normal RyRs fell 5 min after cis 4,49-DTDP was added,
induced three- to fourfold increases in average I9 (Fig. 3),
without any initial activation. There was no further change
after 2.5–3.5 min, while ethanol alone caused an approxi-
inactivityinthesesevenchannelswhen2mMcisDTTwas
mately twofold increase in I9 after ;2.5 min (Table 1).
added, suggesting that the disulfide formed upon exposure
RyR activityreturnedtocontrollevelswithin1minafter
MH to4,49-DTDP(- S-Sr,where-Sriscontributedeitherbythe
10mMDTTwasaddedtothecissideofchannelsactivated a
reactive disulfide or by the channel protein) was reduced
bycisDTNB(fouroffour)ortransDTNB(threeofthree)
duringexposureto4,49-DTDPandhencethattheaffinityof
or after 2 mM cis DTT was added to cis 4,49-DTDP-
- SHfor4,49-DTDPinthesechannelswaslessthannormal.
activated channels (five of five). The similar results with a
normal RyRs and RyR show that the Arg615-to-Cys615
MH
substitution in MH does not alter the ability of sulfhydryl-
Single-channel properties of RyR and RyR
specific reagents to activate RyRs by oxidizing - SH. The MH
a
differencesbetweenRyR andnormalRyRsintherateof There was no significant difference between normal RyR
MH
activation by cis DTNB could be explained by structural (n548)andRyR (n545)inthesteady-stateparameter
MH
changes that reduce the accessibility of - SH in RyR . values for P , F , T , and T measured over 30-s periods
a MH o o o c
(Table 2). The two types of channel showed similar modes
of activity, transitions between modes and responses to
InhibitionofRyR
MH voltage pulses to 140 mV (Fig. 4). Predominant modes
RyR channels were inactivated after 458 6 30 s expo- wereeitherlowactivityorhighactivity,unalteredafterthe
MH
sure to 1 mM cis 4,49-DTDP (added with 1% ethanol, n 5 voltagepulse(Fig.4,IandII)orvoltage-activatedincrease
10),andnineofthe10channelswerereactivated160630s in activity immediately after the voltage pulse, which then
after adding 2 mM DTT. Similarly, 1 mM cis 4,49-DTDP decayed after 10–20 s to a lower level that was also seen
added alone (in cis solution) abolished RyR activity in before the voltage pulse (Fig. 4 III). Strong submaximum
MH
three of six channels after 383 6 156 s, and the three conductanceactivitywasalsoseeninbothnormalRyRsand
TABLE 2 Effectsofoxidizingreagentsandethanolonsingle-channelparametersopenprobability(P ),meanopentime(T ),
0 0
meanclosedtime(T ),andfrequencyofopenings(F )
c 0
P T (ms) T (ms) F (s21)
Condition 0 0 c 0
(n(normal,MH)) Normal MH Normal MH Normal MH Normal MH
Control(8,9) 0.1560.04 0.2060.06 1.1760.07 2.7560.12 8.8261.08 12.4161.03 97619 71617
DTNB(cis) 0.2760.05* 0.4160.06** 5.2660.81** 6.6860.22** 11.2261.22 11.1961.24 62621 64610
Control(6,6) 0.2360.06 0.2560.08 1.6560.10 5.3560.71 6.5560.77 13.2760.98 139631 60614
DTNB(trans) 0.4860.12** 0.5660.09** 4.7260.72* 11.4661.17** 5.7460.97 8.3960.82* 125634 64616
Control(6,6) 0.3260.13 0.1960.06 2.5760.40 2.0260.15 5.6260.70 11.7562.24 88618 95624
DTDP(eth) 0.6760.13* 0.3860.11 13.2363.35** 5.8560.78* 5.8361.58 10.1761.55 74624 73618
Control(4,5) 0.2460.08 0.3860.12 1.8660.26 3.4360.50 6.4360.75 6.6061.18 125618 113628
Ethanol(1%) 0.4460.10* 0.4960.12 4.1060.69* 4.1360.43* 4.6560.57 4.5260.57 118622 119614
Control(7,5) 0.1760.06 0.2060.07 1.2060.19 1.0560.57 7.6561.26 4.1561.81 72617 78631
DTDP(H O) 0.4260.11* 0.4060.09* 8.3262.37** 2.5961.06 9.8462.78 6.8768.50 50617 86626
2
DTNBand4,49-DTDP(DTDP)wereaddedtoafinalconcentrationof1mM.Resultsaregivenasmean61SEM.
Significantdifferencesbetweenparametersundercontrolconditionsandafteradditionofthereagentareindicatedby:*p,0.05.**p,0.01.
DTDP(ethanol),DTDPdissolvedinethanol.
DTDP(HO),DTDPdissolvedincissolution.
2
Haarmannetal. OxidationandReductionofSkeletalRyRs 3015
withanincreaseinmeanclosedtime(T )insomechannels
c
andadecreaseinT inothers,sothatthechangesinaverage
c
F and P were not significant (Table 2).
o o
DTT at 10 mM reversed the effects of DTNB, and at 2
mMreversedtheeffectsof4,49-DTDPinnormalRyRsand
RyR .Datawerepooledfromthefewchannelsthatwere
MH
suitable for analysis and were exposed first to an oxidizing
reagent and then to DTT. I9, P , and T fell significantly
o o
after DTT was added to the oxidation-activated channels
(Fig.5).ThesimilaractionsofcisDTNB,transDTNB,and
cis 4,49-DTDP on the single-channel parameters during
activation and the similar reversal of these actions by DTT
providefurtherevidencethat oneclassof cysteineresidues
(- SH) is oxidized in each of the three situations.
a
The average open channel conductance was higher after
oxidation,being0.5060.05ofthemaximumconductance
beforeand0.5760.09afteroxidationinnormalRyRs(n5
25),or0.3760.05beforeand0.4960.06afterinRyR
MH
(n524,pooleddataforcisDTNB,transDTNB,andcis4,
49-DTDP), although the increase was significant only in
RyR . This suggests that there was an increased fraction
MH
of openings to the maximum conductance and to higher
submaximum conductance levels after oxidation-induced
activation.
Effects of DTNB and 4,4*-DTDP on open and
closed time distributions
Open times for normal RyR and RyR fell into two
MH
exponentialcomponentsundercontrolconditionsandthree
exponential components after oxidation-induced activation
(Figs. 6 and 7). Closed times for both normal RyRs and
RyR channels fell into three exponential components
MH
under control conditions and after oxidiation-induced acti-
vation. Data for normal RyRs and RyR were combined
FIGURE 4 Modesofactivityandopeningstosubmaximumconductance MH
(Fig.7)becausetherewere noconsistentdifferences inthe
levels in normal RyRs and RyR . (A and B) Examples of channels
MH
showinglowactivitybeforeandafteravoltagepulseto140mV(I),high averagetimeconstantsorprobabilityofeventsbetweenthe
activitybeforeandafterapulseto140mV(II),orlowactivitybeforeand two channel types. Under control conditions and during
highactivityafterthevoltagepulse(III).(A)NormalRyRs.(B)RyRMH.(C oxidation-induced activation, the shortest open time con-
andD)AsinglenormalRyRandsingleRyRMH,respectively,bothshow- stant,t ,was;1–2ms,andthesecondtimeconstant,t ,
inglongopeningstoasubmaximumconductancelevel.Thezerocurrent o1 o2
level is indicated by solid lines, and the maximum open conductance is was7–13ms.Thelongesttimeconstant,to3,wasseenonly
shownbydottedlines. in “activated” channels; it was 50–100 ms and contained
,10% of openings. The increase in T was largely due to
o
the appearance of t .
o3
Closed time constants under control and oxidation-in-
RyR (Fig. 4, C and D), giving an average open channel
condMucHtancethatwas0.5060.05ofthemaximumconduc- duced activation conditions were tc1 (2–4 ms), tc2 (9–23
tance in normal RyRs or 0.37 6 0.09 in RyRMH. cmosm),poanndenttc3(t(9)0–co4n8t0ainmesd) v(Feriygs.fe6waenvden7t)s. aTnhde dloidngneostt
c3
change in any consistent way during oxidation-induced
Effectsofoxidizingreagentsandethanolonsingle-channel
activation. The similar open and closed time distributions
parametersofnormalRyRsandRyRMH with cis DTNB, trans DTNB, and cis 4,49-DTDP are con-
Theoxidizingreagentsincreasedmeanopentime(T )inall sistent with the hypothesis that the same cysteine residues
o
normal RyRs and all RyR channels, with significant are oxidized with each reagent. An additional fourth long
MH
increases in average T under most conditions (Table 2). open time constant in cardiac RyRs exposed to cis 4,49-
o
Theeffectonotheraspectsofchannelactivitywascomplex, DTDP (Eager et al., 1999) was not seen in skeletal RyRs.
3016 BiophysicalJournal Volume77 December1999
FIGURE 6 Effectof1mMcisDTNBonopenandclosedtimedistri-
butions for a single RyR . Open and closed times are collected into
MH
loggedbins,andthesquarerootoftherelativefrequencyofevents(f1/2)is
plottedagainstthelogarithmoftheopenorclosedtimesinms.(A)Open
timedistribution.(B)Closedtimedistribution.InAandB,distributionsare
shownforcontroldata(I)andduringactivationby1mMcisDTNB(II).
Thecontinuouslinesineachgraphshowthemultipleexponentialfunction
fitted to the data, and the broken lines show the individual exponential
components(2inAIand3ineachofAIIandBI&II).
mM DTNB or 1 mM 4,49-DTDP was added to the cis
chamber,97%ofchannelsadoptedburstbehavior,inaddi-
tion to the increase in channel open time (Table 3). Only
59% of normal RyRs or RyR had burst behavior with
MH
trans DTNB-induced activation, or 45% during activation
with cis or trans DTT (10 mM, Results below) or with
ethanol (Table 3). The stabilization of burst behavior by
FIGURE 5 EffectsofDTNB(cisortrans)orcis4,49-DTDPonaverage
oxidizing reagents in the cis chamber was not reversed by
channelparametersandreversaloftheeffectsbyDTT.Averagerelative
changesareshownforasubsetofchannelsthatwereexposedfirsttoan DTT.Burstingbehaviorremainedin94%ofchannelswhen
oxidizing reagent (light gray bins) and then to DTT (dark gray bins). 10mMDTTwasaddedtothecisortransbathafter1mM
Pooled data from normal RyRs and RyRMH are shown. Channels were cisDTNB,andremainedin50%ofchannelswhen10mM
exposedto(A)1mMcisDTNBandthen10mMcisDTT(n56),(B)1
DTT was added to thetrans bath after 1 mM trans DTNB.
mM trans DTNB and then 10 mM trans DTT (n 5 4); (C) 1 mM cis
4,49-DTDP(addedincissolution)andthen2mMDTT(n54).Channels This effect of oxidizing reagents on burst activity sug-
wereexposedtoDTT2–3minafterexposuretoDTNBor4,49-DTDP,at gested that DTNB and 4,49-DTDP modified a site on the
timeswhenchannelactivitywashigh.Single-channelparametersshown cytoplasmicsideofthechannelthatalterschannelgatingto
are mean current (I9), open probability (Po), mean open time (To), fre- stabilizeburstingbehavior.Itisunclearwhethertheeffectis
quencyofopening(F ),andmeanclosedtime(T).Thebinsshowmean
o c dueto-SHoxidationoraninteractionbetweentheoxidizing
values and the vertical lines indicate 11 SEM. Asterisks indicate the
reagents and other sites on the channel, because the effect
significanceofdifferencesoftestdatafromcontrol(duringactivation)or
fromtheactivatedlevel(aftertheadditionofDTT).*p,0.05;**p, was not reversed by DTT.
0.01.
Further evidence that - SH is located in the
a
An effect of oxidizing reagents on burst behavior transmembrane domain
Channelopeningsin20–50%ofnormalorRyR channels Ifthehypothesisthat- SHislocatedinthetransmembrane
MH a
under control conditions was continuous, while activity in domain and is accessible from either side of the bilayer is
the other channels was clustered into bursts, separated by correct,thenactivationbyDTNBononesideofthebilayer
closures of 0.5–60 s (not included in Figs. 6 and 7). Con- shouldbereversedbyaddingDTTtotheoppositechamber.
tinuous or burst activity was observed in each of the three Inthisexperiment,fiveofsixRyRchannelswereactivated
modes (high, low, or voltage-activated activity) defined in whenDTNBwasaddedtothecischamber,andactivityfell
the text description of Fig. 4 (I–III) above. When either 1 in five of the channels when DTT was added to the trans
Haarmannetal. OxidationandReductionofSkeletalRyRs 3017
FIGURE 7 Effectsofoxidizingreagentsontheaverage
timeconstantsandfractionofeventsobtainedfrommul-
tiple exponential fits to the open and closed time distri-
butions(e.g.,Fig.6).Theprobabilityofeventsfallinginto
eachtimeconstant(p)isplottedagainstthelogarithmof
thetimeconstantinms(log[opentime,ms]orlog[closed
time,ms]).Theaveragevaluesareforcombineddatafrom
normalRyRsandRyR .(A)Opentimeconstants.(B)
MH
Closedtimeconstants.InAandBdataareshownbefore
andaftertheadditionof1mMcisDTNB(I);1mMtrans
DTNB(II);1mMcis4,49-DTDPwith1%ethanolorcis
1%ethanolalone(III);1mMcis4,49-DTDP(IV).Aver-
agevaluesareshownforcontrolconditons(f,(cid:141))andin
thepresenceoftheoxidizingreagent(M)orethanol((cid:131),
AIIIandBIIIonly).
chamber. Conversely, one of three other channels was ac- mal RyRs and three of three RyR . Data from normal
MH
tivated when DTNB was added to the trans chamber, and RyRsandRyR iscombinedinFig.8.ThetwoRyRsnot
MH
activityfellinthatchannelwhenDTTwasaddedtothecis activated by trans DTT were not included, because their
chamber. controlactivitywashigh(I9of4.0and6.8pA)andoutside
therangeof0.2–1.2pAintheotherfivechannels.Average
I9 increased significantly with 10 mM DTT (cis or trans)
Evidence for a modified thiol in the
and then fell significantly after DTNB addition to the op-
transmembrane domain
posite chamber (Fig. 9). Interestingly, activity in the two
DTTat2mM,addedintheabsenceofanoxidizingreagent, channels that were not activated by cis DTT fell to lower
did not significantly alter RyR activity. The mean current, levels when DTNB was added to the trans chamber, with
normalized to control, was 3.6 6 2.8 in five normal RyRs similar approximately sixfold reductions in I9. These chan-
after 2 mM DTT addition and 2.7 6 0.4 in five RyR nels may have already been in a reduced state before the
MH
channels. On the other hand, 10 mM DTT significantly addition of DTT.
activatednormalRyRsandRyR whenaddedtoeitherthe Theseresultssuggestthataclassofmodifiedthiol(- S-
MH ab
cis or trans chamber in the absence of added oxidizing R-, where R is either a protein S if the modified thiol is a
reagent;activitythenfelltowardcontrollevelswhen1mM disulfide,oraNifthemodifiedgroupisnitrosylated;Xuet
DTNBwasaddedtotheoppositesideofthebilayer(Fig.8). al.,1998)isavailableto10mMDTTfromeitherthecisor
CisDTT(10mM)activatedfiveoffiveRyRs(twonormal; trans chamber and that the reduced - SH can be oxidized
ab
three RyR ), while trans DTT activated two of four nor- byDTNBfromtheoppositesideofthebilayer.Theacces-
MH
3018 BiophysicalJournal Volume77 December1999
TABLE 3 EffectsofoxidizingreagentsandethanolonburstbehaviorofnormalRyRsandRyR
MH
Before3After Before3After Before3After Before3After
Continuous3 Continuous3 Bursting3 Bursting3
Continuous Bursting Bursting Continuous
DTNB(cis)
NormalRyR 0 8 10 0
RyR 0 2 16 0
MH
DTNB(trans)
NormalRyR 8 0 4 1
RyR 3 0 15 1
MH
4,49-DTDP(eth)(cis)
NormalRyR 0 6 9 1
RyR 0 1 13 0
MH
4,49-DTDP(HO)(cis)
2
NormalRyR 2 3 18 0
RyR 0 0 17 0
MH
Ethanol(1%)(cis)
NormalRyR 1 1 7 1
RyR 0 1 13 1
MH
Oxidizingreagentswereaddedtoafinalconcentrationof1mM.Thenumbersofchannelsareshownwithcontinuousorburstingactivitybeforeandafter
additionoftheagentslistedontheleft-handside,fornormalRyRsandRyR .
MH
4,49-DTDP(eth),4,49-DTDPdissolvedinethanol.
4,49-DTDP(HO),4,49-DTDPdissolvedincissolution.
2
sibilityfromeithersideofthebilayerindicatesthat- S-R- 4,49-DTDP activated and then inhibited RyRs from the
ab
and - SH are located in the transmembrane domain. cytoplasmicsolution.Activationbybothreagentsandinhi-
ab
bition by 4,49-DTDP were reversed by DTT and thus are
duetosulfhydryloxidation.Theresultscanbeexplainedby
“Control-like” channel activity is retained in the
oxidation of two classes of sulfhydryl, - SH in the trans-
presence of 1 mM DTNB plus 10 mM DTT a
membrane domain for activation or -SH in a hydrophobic
i
Channel activity returned to “control-like” levels when 10 environmentforinhibition.Separate- SHand-SHresidues
a i
mMDTTand1mMDTNBwerepresentoneitherthesame have also been postulated for cardiac RyRs (Eager and
or opposite sides of the bilayer. This was confirmed in Dulhunty,1998,1999).Additionalnovelfindingswerethat
furtherexperiments,inwhichthe“control-like”activitywas 1)additionoftheoxidizingreagentstothecissideofRyRs
maintainedif10mMDTTwaspresentonbothsidesofthe stabilized bursting channel activity, suggesting that cyto-
channel, with 1 mM DTNB on one side only (five of six plasmic residues regulate burst activity; 2) addition of 10
experiments with cis DTNB, or three of three with trans mM DTT to either side of the channel caused activation,
DTNB). Average I9 measured in four of the channels with whichwasreversedwhenDTNBwasaddedtotheopposite
cis DTNB or two of the channels with trans DTNB was side, suggesting that a modified thiol - S-R- in the trans-
ab
22.19 6 0.32 pA. “Control-like” activity was retained membrane domain normally inhibits activity; and 3) “con-
when10mMDTTwasremovedfromonechamber,leaving trol-like”channelactivitywasmaintainedinthepresenceof
10 mM DTT and 1 mM DTNB in the cis chamber in two 1 mM DTNB and 10 mM DTT. Finally, the effects of the
cases,orinthetranschamberinathirdcase(I9521.336 oxidizingreagentsonRyRsfromnormalandMHpigswere
0.51 pA). similar.
The assertion that channel activity in the presence of 10
mM DTT and 1 mM DTNB was “control-like” was sup-
Activation of skeletal RyRs by reactive disulfides
ported by a final experiment (n 5 2) in which the trans
chamberinitiallycontained1mMDTNBplus10mMDTT ActivationofskeletalRyRsbyDTNBand4,49-DTDPcon-
andthecischambercontained1mMDTNB(I9520.976 firms previous reports on skeletal RyRs (Nagura et al.,
0.39 pA). The trans chamber was perfused with normal 1988; Marengo et al., 1998; Zable et al., 1997). Similar
trans solution, leaving 1 mM DTNB in the cis solution. In activation,withalongtimeconstantcomponentintroduced
both cases, RyR activity increased after perfusion, as it into the open time distribution, is seen in cardiac RyRs
usuallydidwhenDTNBwaspresentaloneinthecischam- exposed to 4,49-DTDP or thimerosal. An increase in open
ber (I9 5 25.50 6 0.33 pA). frequency is also seen in cardiac RyRs. A fourth long time
constantcomponentintheopentimedistributionofcardiac
RyRs oxidized by 4,49-DTDP was not seen in skeletal
DISCUSSION
RyRs, suggesting that the - *SH class of sulfhydryl, postu-
a
WefoundthatDTNBactivatedskeletalmuscleRyRsfrom lated for the cardiac RyR (Eager and Dulhunty, 1998),
eitherthecytoplasmicorluminalsideofthechannel,while either is not present or is not available for oxidation in
Haarmannetal. OxidationandReductionofSkeletalRyRs 3019
FIGURE 9 AverageeffectsonI9ofactivationby10mMDTTaddedto
thecisortranschamberandreversaloftheactivationwhen1mMDTNB
isaddedtotheoppositechamber.Thecross-hatchedbinsshowresultsfor
cisDTTfollowedbytransDTNB.Thestippledbinsshowdataresultsfor
trans DTT followed by cis DTNB. The average I9 (in pA) is given for
control conditions (control), after the addition of DTT (DTT), and then
aftertheadditionofDTNB(DTNB).Asterisksindicatethesignificanceof
the difference between DTT and control, or between DTNB and DTT.
*p,0.05;**p,0.01.
ofGSSGtoactivateskeletalRyRsfromtheluminalsolution
(Zable et al., 1997) might have been due to the weak
oxidizing ability of GSSG.
What do similar changes to reagents added to
either side of the RyR mean in terms of the
location of target residues?
The ability of DTNB to activate RyRs from the luminal or
cytoplasmic side and the reversal of activation by 10 mM
DTT added to the opposite side of the channel suggest that
- SHisaccessibletoDTNBandDTTfromthecytoplasmic
a
and luminal solutions. This accessibility to water-soluble
reagentscouldsuggestthattargetresiduesarelocatedinthe
channelpore.However,DTTwithapK of9.0–10(Shaked
a
et al., 1980) would be largely uncharged at pH 7.4 and
would rapidly partition into the membrane. Similarly,
1–10% of DTNB with a pK of 5–6 (Houk et al., 1987)
a
FIGURE 8 Activation of normal RyRs and RyR by 10 mM DTT would enter the membrane. Thus the water-soluble agents
MH
added to the cis or trans chamber without oxidizing reagent, and the couldaccessresidueslocatedinthetransmembranedomain,
reversal of activation by the addition of 1 mM DTNB to the opposite notinthepore,althoughactivatingresiduesinatransmem-
chamber. (A and C) Normal RyRs. (B and D) RyRMH. Records in A–D brane,ratherthanporelocation,wouldseeonly10–100mM
show control activity (I); activity with 10 mM DTT (II); activity after
DTNB.Thisisnotanunreasonable[DTNB]foractivation,
additionof1mMDTNB(III).Drugadditionswere(AandB)cisDTTthen
transDTNBand(CandD)transDTTthencisDTNB.Thezerocurrent because DTNB is a strong oxidizing reagent (above), and
level is indicated by solid lines, and the maximum open conductance is NO at ;40 nM nitrosylates thiol groups and activates
shown by broken lines. Bilayers in A, B, and D contained one active skeletal RyRs (Hart and Dulhunty, unpublished observa-
channel,whilethebilayerinCcontainedthreeactivechannel.Thethree tions), while cardiac RyRs are activated by 100 mM 4,49-
brokenlinesinCindicatethemaximumopenconductancewithone,two,
DTDP (Eager et al., 1997).
orthreechannelsopeninthebilayer.
It is unlikely that the reagents, either crossing the mem-
brane or passing through the pore, could have targeted
skeletalRyRs.Thefactthatreversalofactivationby1mM residues located on the opposite side of the channel and
DTNB required 10 mM DTT, while reversal of activation remote from the membrane, because dilution in the large
by 4,49-DTDP or thimerosal requires 2 mM DTT (results volume of solution would mean that it would take a long
above and Eager et al., 1999), suggests that DTNB has time for their concentrations to increase to active levels.
stronger oxidative properties than 4,49-DTDP. The failure Thus activation with DTNB or recovery with DTT would