Molecular Identification of a Mechanosensitive Channel in Archaea

12 Pages·2000·0.75 MB·English

BiophysicalJournal Volume80 January2001 229–240 229
Molecular Identification of a Mechanosensitive Channel in Archaea
Anna Kloda and Boris Martinac
DepartmentofPharmacology,QEIIMedicalCenter,TheUniversityofWesternAustralia,NedlandsWA6907,Australia
ABSTRACT TheTM1domainofthelargeconductancemechanosensitive(MS)channelofEscherichiacoliwasusedasa
geneticprobetosearchthegenomicdatabaseofthearchaeonMethanoccoccusjannashiiforMscLhomologs.Wereportthat
the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL.
Moreover,MJ0170wasfoundtobeaconservedhomologofMscS,thesecondtypeofE.coliMSchannelencodedbythe
yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly
resembledthechargedC-terminalaminoacidclusterpresentinEco-MscL(RKKEE).WeclonedandexpressedMJ0170in
E.coli,whichwhenreconstitutedintoliposomesorexpressedinthecellmembraneofgiantE.colispheroplasts,exhibited
similaractivitytothebacterialMSchannels.OurstudysuggeststhattheM.jannashiiMSchannelanditshomologsevolved
as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved
structuralmotifamongprokaryoticMSchannels.
INTRODUCTION
Traditionallyionchannelshavebeenstudiedinthecontext (Bult et al., 1996) for MscL homologs in Archaea. We
of neurophysiology and within eukaryotic metazoan prepa- identified the hypothetical protein (MJ0170) as a putative
rations (Hille, 1992). With the advent of the patch-clamp MSchannelwithahighdegreeofhomologytoEco-MscL.
recording technique (Hamill et al., 1981), studies of ion OnalignmentofthesequencesofMJ0170andtherecently
channels in various microbes including prokaryotes gained cloned E. coli MS channel of the small conductance (Eco-
momentum. Mechanosensitive (MS) ion channels have MscS) (Levina et al., 1999), we found a high degree of
beenextensivelystudiedinbothGram-negativeandGram- homology between these two proteins. This approach has
positive bacteria (Zoratti and Ghazi, 1993; Martinac et al., allowed recognizing and establishing the mutual relation-
1992;Martinac,1993;Blountetal.,1999).Theexistenceof ship and common evolutionary origin of the new family of
MS ion channels in cell membranes of Archaea, the third prokaryotic (bacterial and archaeal) MS channels.
domain of the universal phylogenetic tree (Woese, 1994;
SteinandSimon,1996;Pace,1997)wasfirstdocumentedin
thearchaebacteriumHaloferaxvolcanii(formerlyHalobac-
MATERIALS AND METHODS
terium volcanii) (Le Dain et al., 1998). As a distinct group
of prokaryotic microorganisms archaebacteria comprise Bacterial strains and culture conditions
several different families of cells adapted to extreme envi-
E.colicellsharboringtheAMJBZ56cloneencodingtheMJ0170protein
ronmentssuchassuper-hotoceanhydrothermalventsorthe wereobtainedfromAmericanTypeCultureCollection(Rockville,MD).E.
high salt concentrations found in the Dead Sea (Barinaga, colistrainM15(pREP4::kan)(Qiagen,Chatsworth,CA)wasusedasahost
1994). The existence of MS channels in archaeal and bac- for a recombinant plasmid harboring the MJ0170 gene. E. coli strain
MJF465ofthefollowinggenotypeFrag1,DmscL::Cm,DyggB,DkefA::kan
terialcellmembranessuggeststhatthisclassofionchannels
(Levinaetal.,1999)wasusedforproteinexpressionandelectrophysiol-
mighthaveappearedveryearlyduringtheevolutionoflife
ogy.Strainsweregrownat37°CinLuria-Bertanibroth(LB)containing
on Earth (Garcia-An˜overnos and Corey, 1997; Martinac, 10g/L Bacto-tryptone, 5g/L yeast extract, and 5g/L NaCl supplemented
1999). withampicillin(100mg/ml),chloramphenicol(20mg/ml),andkanamycin
TheTM1domainofthebacterialMSionchanneloflarge (25mg/ml),accordingtotheselectionrequirements.Thegeneexpression
conductance(MscL)ishighlyconservedamongGram-neg- wasinducedwithisopropyl-1-thio-b-D-galactopyranoside(IPTG)oncethe
cellculturereachedmid-logphase(OD of;0.6).
ative and Gram-positive bacteria (Sukharev et al., 1997; 600
Moe et al., 1998; Batiza et al., 1999; Spencer et al., 1999;
Oakleyetal.,1999).InthisstudyweusedtheTM1domain
Cloning and protein purification
ofMscLofEscherichiacoli(Eco-MscL)asageneticprobe
toscreenthegenomicdatabaseofMethanococcusjannashii TheTM1domainofEco-MscLwasusedasageneticprobetosearchthe
M. jannaschii genomic database for a putative MscL homolog. The se-
quence alignment was generated using SIM Alignment Tool for Protein
SequencesavailableattheExPasyMolecularBiologyserver.Thealign-
Receivedforpublication21July2000andinfinalform11October2000.
ment that produced the highest score was used to generate either the
AddressreprintrequeststoDr.BorisMartinac,DepartmentofPharmacol-
Kyte-Doolittlehydropathyplotorthedensealignmentsurface(DAS)plot
ogy,QEIIMedicalCenter,TheUniversityofWesternAustralia,Nedlands,
for transmembrane segments prediction (ExPasy) to ensure the newly
WA6907,Australia.Tel.:[phone];Fax:[phone];E-mail:
identified protein had the properties of a membrane protein. The entire
[email protected].
openreadingframeofMJ0170wasamplifiedbypolymerasechainreaction
© 2001bytheBiophysicalSociety (PCR)usingtheAMJBZ56cloneasatemplateandclonedintopQE-32
0006-3495/01/01/229/12 $2.00 expression vector (Qiagen) as a BamHI-SalI fragment using standard
230 KlodaandMartinac
cloningprocedures(Sambrooketal.,1989).The6xHisMJ0170recombi- distributionfunctionoftheformNP 5NP [11expa(p 2p)]21,
o omax 1/2
nantproteinwaspurifiedaspreviouslydescribed(Sukharevetal.,1999). whereNistheunknownnumberofchannelsinthepatch,P istheopen
o
probability, P is the maximum open probability, p is the negative
omax
pressureappliedtothepatchpipette,p isthepressureatwhichtheopen
Liposome preparation, protein reconstitution, and 1/2
probabilityis0.5,andaisthechannelsensitivitytopressure.Thevalues
spheroplast preparation forp and1/aofMscSestimatedingiantspheroplastsofE.coliwere
1/2
36623mmHgand561mmHg(n59),respectively(B.Martinac,
The MJ0170 protein was reconstituted into liposomes according to the
unpublished results). The single-channel open probability was estimated
methods described previously for MS ion channels in E. coli and H.
fromthetotalcurrentdividedbythesingle-channelcurrentgivingNP and
volcanii(Delcouretal.,1989;Sukharevetal.,1993;Ha¨seetal.,1995;Le o
divided by the maximum number of channels observed in the patch. By
Dain et al., 1998). Bilayer blisters of 50–100 mm in diameter, which usingatwo-stateBoltzmannmodelwiththechangeofareatDAbeingthe
appearedafterincubationoftheproteoliposomesintherecordingsolution
dominantenergyterm(Sukharevetal.,1999),itfollowsaccordingtothe
(Delcouretal.,1989),wereexaminedusingthepatch-clamptechnique. modelofHowardetal.(1988)thatthefreeenergyDGisalinearfunction
Forspheroplastpreparation,bacterialcellsAMJ465harboringtheplas- ofmembranetensiont;i.e.,DG5tDA2DG,whereDG isthedifference
mid pQE-32MJ0170 were cultured to mid-log phase as described for o o
infreeenergybetweentheclosedandopenconformationsofthechannel
protein purification experiments and induced with IPTG for 30 min. intheabsenceoftheexternallyappliedmembranetensionandDAisthe
Spheroplasts were then prepared according to the previously described
differenceinmembraneareaoccupiedbyanopenandclosedchannelata
method(Martinacetal.,1987). givenmembranetension,whereastDAistheworkrequiredtokeepaMS
channelopenbyexternalmechanicalforceattheopenprobabilityof0,
Cell-free expression P ,1.TheBoltzmannfunctionfortheopenprobabilityofasingleMS
o
channelcanbewrittenasP /(12P)5exp[a(p2p )]5exp[(tDA2
o o 1/2
The TNT Quick Coupled Transcription/Translation system was used for DG)/kT].Becausemembranetensiontisnearlyproportionaltothepres-
o
transcriptionandtranslationofMJ0170genecloneddownstreamfromSP6 surewithintherangeappliedtothepatchpipetteinthisstudy,itiswell
RNApolymerasepromoterofthepSP64poly-Avector.Forproteinlabel- approximatedbyamodifiedformoftheLaplace’slaw,suchthatt2t 5
1/2
ing, 25-ml reactions containing 0.5 mg of either pSP64-polyA MJ0170 (p2p )(r/2),whereristheradiusofcurvatureoftheliposomemembrane
1/2
constructoremptyvectorweresetupwith40mlofTNTmixofreticu- patchunderexternalnegativepressurepappliedtothepatchpipette.Thus,it
locyte lysate, SP6 polymerase, a complement of amino acids without followsthatwhentheopenprobabilityP 50.5(i.e.,p5p andt5t )the
o 1/2 1/2
methionine, and Rnasin (RNAse inhibitor). The reactions were supple- free energy difference DG 5 0. Consequently, t 5 DG/DA and p 5
1/2 0 1/2
mented with 2 ml of [35S]methionine (10 mCi/ml) and 2.5 ml of canine 2DG/rDA,whereasa5rDA/2kT.
0
pancreatic microsomes (Sukharev et al., 1994). For details see also Pro-
mega Technical Bulletin 126 (Promega, Madison, WI). After 2 h of
incubation at 30°C the reactions were heated at 100°C for 5 min in the RESULTS
presence of sample buffer (6% 32 b-mercaptoethanol, 3% SDS, 0.3%
bromophenol blue, and 1% glycerol) and separated on 12% SDS-PAGE Multiple sequence alignment and phylogeny of
gel.Thegelwasfixedinasolutioncontaining10%aceticacidplus30% prokaryotic MS channels
methanol,driedundervacuum,andexposedtox-rayfilm.Forfunctional
assayofchannelactivity,50-mlreactionscontainingeitherpSP64-polyA WeconsideredtwotypesofalignmentsofMJ0170(referred
MJ0170constructoremptypSP64-polyAvectorweresetupwithafull toasMscMJ),thelocalalignmentagainstE.coliMscL(Fig.
complementofunlabeledaminoacidsunderconditionsdescribedabove.
1 A) and the global alignment against identified MscL and
MicrosomalmembraneswerewashedinKClbuffer(200mMKCl,10mM
HEPES-KOH,pH7.0),andthemembranepelletwasresuspendedin50ml MJ0170homologs(Fig.2A).Thelocalalignmentidentified
of liposome suspension containing 2 mg of phosphatidylcholine supple- MscL-like motifs TM1, TM2, and TM1-loop, which were
mentedwith10%cholesterolin10mMMOPSbuffer,pH7.2.Dropletsof preserved within the first, the second, and the third helical
mixed suspension were subjected to a dehydration-rehydration cycle de- domains of MJ0170, respectively, with the following
scribedpreviously(Delcouretal.,1989;Ha¨seetal.,1995),andproteoli-
scores: 1) 38.5% identity in the stretch of 26 residues
posomes were examined for the channel activity using the patch-clamp
corresponding to the TM1 transmembrane helix of Eco-
technique.
MscL and the first putative membrane-spanning domain of
MJ0170,2)31.8%identityinthe22-residueoverlapcorre-
Electrophysiological recording
spondingtomostoftheTM2transmembranehelixofEco-
Single-channel currents were filtered at 2 kHz, digitized at 5 kHz, and MscL and the second putative membrane-spanning domain
analyzed using pCLAMP6 data acquisition and analysis software (Axon of MJ0170, and 3) 40% identity in 20 amino acid residues
Instruments, Foster City, CA). Current recordings were viewed by the
encompassingasectionoftheTM1helixplustheperiplas-
AxoscopeforWindowsprogram(AxonInstruments).Currentamplitudes
mic loop of Eco-MscL and a section of the third putative
weredeterminedbymeasuringthedifferencebetweenthecursoralignedat
peak and baseline currents. Channel conductance was estimated from transmembrane domain of MJ0170. Furthermore, the
current voltage plots. Suction applied to the patch-clamp pipette was MJ0170 amino acid sequence could be aligned against the
measured by the piezoelectric pressure transducer (Omega Engineering, sequence of the YggB protein underlying the activity of
Stamford,CT).
Eco-MscS, the bacterial MS channel of small conductance
(Levinaetal.,1999).Thisalignmentexhibited28%identity
Estimate of the MS channel free energy of
in the overlapping 226 residues of the YggB sequence
activation from Boltzmann distribution
encompassing the TM1-periplasmic loop region of MscL
and the third putative transmembrane helix and C-terminal
Open probability of the MscMJ channels plotted against the negative
pressure(suction)appliedtothepatchpipettewasfittedtoaBoltzmann portion of MJ0170. The alignment demonstrated that the
BiophysicalJournal80(1)229–240
MechanosensitiveChannelofM.jannaschii 231
FIGURE 1 Structural properties of the MJ0170 (MscMJ) mechanosensitive channel protein. (A) Regions of homology between MJ0170, MscL, and
YggBproteinsequences.TheidenticalresiduesfoundinboththeMscLandMJ0170aminoacidsequencesaremarkedwithanasterisk,whereasthose
commontotheYggBandMJ0170sequencesaremarkedwithfilledcircles.TM1andTM2MscLtransmembranedomainsthatmatchMJ0170homolog
sequences are boxed. (B) Hydropathy profile and predicted a-helical secondary structure (black bars) of MJ0170. Positive numbers indicate relative
hydrophobicityintheplot.(C)HelicalwheelrepresentationoftheputativefirsttransmembranedomainTM1ofMJ0170revealsitsamphipathiccharacter.
Chargedresiduesareboxed.Thestartofthehelixismarkedwithanasterisk.
BiophysicalJournal80(1)229–240
232 KlodaandMartinac
BiophysicalJournal80(1)229–240
MechanosensitiveChannelofM.jannaschii 233
first, part of the second, and the third membrane-spanning the third branch. The proteins on the branch with no MscL
domain of MJ0170 shared high homology with MscL, areapproximatelyfourtimesthesizeofMJ0170homologs
whereas the large portion of the MJ0170 sequence starting and have preserved MJ0170-like rather than the MscL-like
at the third transmembrane helix and including most of the structural motif indicating their more recent origin.
C-terminus shared high homology with the YggB protein.
Inaddition,aclusterofchargedresiduesisconservedinthe
C-terminal domains of all three proteins, MscL (RKKEE),
Secondary structure analysis
YggB (RIKRE), and MJ0170 (KIKEE) (Fig. 1 A). This
finding is of potential significance, as MscL activity was Hydropathy plot analysis combined with the secondary
abolished in a mutant having 33 C-terminal residues that structure prediction (Fig. 1 B) indicated that MJ0170 had
includedthechargedclusterRKKEEdeleted(Blountetal., threemembrane-spanningdomainsfollowedbyaverylarge
1996; Ha¨se et al., 1997). Interestingly, a charged cluster hydrophilic C-terminal tail. Moreover, the helical wheel
withaverysimilarsequenceRVISKKTKEEisalsopresent analysisoftheputativeTM1helixofMJ0170indicatedthat
in the C-terminal domain of the mammalian mechanogated the helix is amphipathic in character (Fig. 1 C) and may
potassium channel TREK-1 (Patel et al., 1998). therefore constitute a structural part of the channel pore.
Multiplesequencealignmentrevealed1)ahighdegreeof Becauseithasbeensuggestedthatglycosylationinthermo-
preservationofallthreehelicaldomainsofMJ0170among philic Archaea serves to stabilize proteins at high tempera-
its homologs (data not shown) and 2) a recognizable con- tures (Voorhorst et al., 1997), we searched for putative
sensuswithinthealignmentofMscMJandMscLhomologs glycosylationsitesinMJ0170usingtheNetOGlyc2.0pro-
(Fig. 2 A). MscL homologs align to the C-terminal portion gram. We identified threonine 283 as a high-probability
of MJ0170 homologs with prominent conservation of the O-glycosylation site, which together with the hydropathy
MscL TM1 motif within the distal portion of the third plot indicated that MJ0170 belonged to the type VI of
transmembrane helix of MJ0170. Glycine residues sepa- membrane proteins having an odd number of membrane-
ratedbythreetofourhydrophobicresiduesformthesigna- spanningheliceswiththeN-terminuslocatedinthecytoplasm
ture sequence of this region among all aligned proteins. andtheC-terminusintheextracellularspace(Reithmeierand
Furthermore, proline and glycine residues at the MscL Deber,1992).
TM1-periplasmic loop interface are highly conserved
among nearly all aligned proteins. Interestingly, it was
demonstrated that proteolytic cleavage of the MscL
Mechanosensitivity of MscMJ
periplasmic loop led to a dramatic increase in channel
pressuresensitivity(Ajouzetal.,2000),possiblyindicating To determine whether MJ0170 is a mechanosensitive pro-
the importance of the conserved residues in controlling the tein we amplified the entire open reading frame of the
channel mechanosensitivity. MJ0170 gene by PCR, cloned it into an expression vector,
The phylogenetic tree of aligned sequences revealed a and expressed it in E. coli as a 6xHis-tagged recombinant
commonoriginofMSchannels(Fig.2B)incellsbelonging protein(seeMaterialsandMethods).TheSDS-PAGEanal-
to both prokaryotic (archaeal and bacterial) domains of the ysisshowedthatMJ0170proteinranasa37-kDaband(Fig.
universaltreeoflife(Fig.2C).Sequencesareclusteredinto 3 A, left). The purified protein was reconstituted into lipo-
three main branches on the basis of their similarity, which somes and found to exhibit MS-channel-type activity char-
reflectstheircommonancestryandevolutionarydivergence acterized by long openings when examined by the patch-
overtime.Twoofthemainbranchesconsistofamixtureof clamptechnique(Fig.3A,right).MJ0170exhibitedsimilar
MscLandMJ0170homologswhereasMscLisabsentfrom MSchannelactivitywhenexpressedandexaminedbypatch
FIGURE 2 PhylogenyofprokaryoticMSchannels.(A)MultiplesequencealignmentofMscMJandMscLhomologs(ClustalX1.8).Thealignment
includestheC-terminalportionofMscMJanditshomologsstartingfromhelix3(markedwithahorizontalblackline)andthecompletesequencesofMscL
homologs.Thecoloredresiduessharesimilaritywiththeconsensus,whichisdefinedbythemaximizedalignmentofallsequences.Yellowrepresents
proline residues, brown represents glycine residues or its divergent substitution with basic residues, green is assigned to polar residues, whereas blue
representshydrophobicresiduesortheirdivergentsubstitutionswithmostlyacidicresidues(seeClustalX4colorindexformoreinformationonthecolor
schemechosenforthisfigure).Highlyconservedresiduesaremarkedwithanasterisk.Themostextensiveconsensussequencebetweenallalignedproteins
ismarkedbythestartofthehelix3ofMscMJ(blackhorizontalline)andTM1domainofMscL(purplehorizontalline).Thearrowpointstoahighly
conservedasparigineresidue(N182)withinthehelix3ofMscMJanditshomologs,whichisreplacedwithlysineinE.coliMscL(K31)andmostofits
homologs.ThesequencesarenumberedandtheirnamesgiveninB.(B)PhylogenetictreeofcompletealignedsequencesofMscMJhomologsandMscL
homologs showing the common ancestry of prokaryotic MS channels. The archaeal homologs of MscMJ are boxed (red). Numbers in parentheses
correspondtothesequencenumberinA.(C)Universalphylogenetictreederivedfromcomparativesequencingof16SribosomalRNA(modifiedfrom
Pace,1997,withpermission).MscMJhomologsarefoundintwomajordomainsoflife:bacteriaandarchaea.Theredarrowspointtoarchaealphylaof
ArchaeoglobusandMethanococcuswithknowngenomicDNAsequences,whichwerefoundtoharborMscMJhomologs.
BiophysicalJournal80(1)229–240
234 KlodaandMartinac
FIGURE 3 Molecular identification of
MscMJ.(A,left)SDS-PAGEofthe6xHis
MJ0170 protein expressed in E. coli.
Lanes1to7indicateproteinpatternbe-
fore(lane1)andafter(lane2)induction
withIPTG,flowthroughaNi-NTAcol-
umn (lane 3), 20 mM imidazole wash
(lane 4), and the first, second, and third
250-mMelutionfraction(lanes5,6,and
7, respectively). (A, right) Activation of
thepurifiedrecombinantMJ0170protein
reconstitutedintoazolectinliposomesby
negativepressureappliedtothepatchpi-
pette. Upper trace shows single-channel
currentfluctuationsrecordedat250-mV
pipettevoltage.Cdenotestheclosedstate
andO denotestheopenstateofnnumber
n
of channels in the particular liposome
patch. Lower trace shows the negative
pressureappliedtothepatchpipette.(B)
Cell-free expression of the MJ0170 pro-
tein. The autoradiogram of the SDS-
PAGEontheleftshowstheexpressionof
the MJ0170 protein band in the lysate
driven by a mj0170-containing vector
(lane 1) or no protein expression in the
controllysatecontainingtheemptyvector
(lane 2). Activities of the MS channels
were recorded in a liposome patch con-
taining a membrane fraction of the ex-
pression lysate at 140-mV pipette volt-
age without applied negative pressure
(lower trace) and at 230 mV with ap-
plied pressure (upper trace). Closed and
openstatesofthechannelsaremarkedC
andO,respectively.
clamp in giant spheroplasts of E. coli (data not shown) conductance,butitcouldbethatinthecell-freesystemthe
(Martinac et al., 1987). channelmighthaveformedanoligomerofalargernumber
In addition, we expressed the MJ0170 protein in vitro in of subunits than in the E. coli membrane. Furthermore,
a cell-free (rabbit reticulocyte lysate) transcription/transla- some difference in the channel conductance might have
tion system. This system proved to be successful with originated from the post-translational modifications of the
heterologousexpressionofMscLgeneandwasreportedto protein, such as glycosylation in the cell-free expression
bedevoidofionchannelactivities(Sukharevetal.,1994)in system (Technical Manual TM231 and Technical Bulletin
accordance with our control experiments that contained 126,Promega).Becausethelargeconductanceeventswere
empty vector. Autoradiographic analysis showed that the observedexclusivelyinthecell-freeexpressionsystem,the
proteinrunsasabandofsimilarsize(Fig.3B,left)tothat remaining analysis in this study was confined to the small
of the protein expressed in the E. coli membrane (Fig. 3 A, conductance events.
left). The MJ0170 protein expressed in the cell-free system When plotted against the negative pressure the open
exhibitedMSchannelactivitywhenreconstitutedintolipo- probability of the MJ0170 MS channel could be fitted to a
somes. Two types of conductances were observed: the Boltzmann distribution function (Fig. 4 A), which demon-
smaller conductance corresponded to that of the protein strated that the MJ0170 channel protein is gated by mem-
expressedinE.coliandthelargerapproximatelythreetimes brane tension. Consequently, we propose to rename the
its size (Fig. 3 B, right). At present, we do not know the hypotheticalproteinMJ0170asMscMJ,formechanosensi-
cause for the appearance of the MS channel with larger tivechannelofM.jannashii.Theresultsfromfourdifferent
BiophysicalJournal80(1)229–240
MechanosensitiveChannelofM.jannaschii 235
FIGURE 4 MechanosensitiveandconductivepropertiesoftheMJ0170MSchannelprotein.(A)TheopenprobabilityoftheMscMJchannelfromfour
differentpatcheswasplottedagainstthenegativepressure(suction)appliedtothepatchpipetteandfittedtoaBoltzmanndistributionfunction.Thechannel
sensitivitytopressure1/avariedbetween9and13mmHgpere-foldchangeinopenprobability,andthepressurerequiredforhalf-activationp (where
1/2
P 50.5)variedbetween50and64mmHg.Single-channelopenprobabilityversusnegativepressurewasobtainedbydividingopenprobabilityofmultiple
o
channels(NP )bythenumberofactivechannelsintheparticularpatch(N)obtainedfromthebestfitoftheplotln[NP /(NP 2NP )].Saturatingdose
o o omax o
responseswereobtainedintwopatches.Intheremainingtwopatches,80%ofthesingle-channelopenprobabilitywasachieved.Thepressuresatwhich
patchlysisoccurredwerenotusedforanalysis.(B)Current-voltagerelationshipforMscMJ.Single-channelconductancecalculatedfromthecurrent-voltage
plotobtainedin200mMKCl,5mMMgCl ,and5mMHEPES(pH7.2)bufferwas270pS.Thereversalpotentialwasobtainedforthesamepatchby
2
fittingasecond-orderpolynomialcurvetothecurrent-voltagerelationacquiredafterreplacingthebathsolutionwiththebuffercontaining600mMKCl,
5mMMgCl,and5mMHEPES(pH7.2)was18mV,whichindicatedthechannelpreferenceforcationsoveranions.Theionicstrengthandosmolarity
2
werebothchangedthreefold.Thereversalpotentialsforchloride(E )andpotassium(E )areindicatedbyarrows.(C)IonicselectivityofMscMJ.Closed
Cl K
symbolsrepresentmonovalentcations:K1(F),Na1(f),Li1((cid:140)),Rb1(l),andCs1((cid:141)).Opensymbolsrepresentdivalentcations:Ba21(E),Mg21(M),
andCa21((cid:130)).Eachdatapointismean6SEofatleastthreeexperiments(n53).(D)CurrenttracesofMscMJrecordedat140-mVpipettevoltagein
differentbufferscontainingeither200mMXClor200mMYCl plus40mMMgCl ,5mMHEPES(pH7.2),whereXandYsymbolizemonovalentand
2 2
divalentcations,respectively.Negativepressuresappliedtoactivatethechannelswereintherangebetween50and100mmHg.
BiophysicalJournal80(1)229–240
236 KlodaandMartinac
patches showed that the sensitivity of MscMJ to pressure TABLE 1 IonselectivityofMscMJ
1/a51162mmHgpere-foldchangeinthechannelopen
Conductance
probability, whereas the pressure required for 50% open Ion (pS) N P /P
X K
probability p 5 57 6 6 mm Hg.
1/2 Li1 16867 3 0.6
Voltage of either sign not only caused an increase in the Na1 16966 3 0.6
channel opening probability but also affected the channel K1 27164 5 1.0
kineticscharacterizedbylongopeningsatvoltages#90mV Rb1 30664 7 1.1
and very brief openings at voltages $90 mV (data not Cs1 30367 7 1.1
Mg21 18462 4 0.7
shown).Itislikelythatcloselyclusteredpositivechargesof
Ba21 18064 6 0.7
fourlysinesandonearginineononesideoftheamphipathic Ca21 25968 5 1.0
TM1 helix (Fig. 1 C) may confer the voltage sensitivity to
P /P (whereX5otherions)isthecalculatedratiooftheconductanceof
the channel. thXechKannel(i.e.,P /P 5g /g )insolutionscontaining200mMofthe
X K X K
particularionXtotheconductancecalculatedfor200mMpotassium.
Ionic selectivity
Next, we also estimated the conductance and selectivity of tionoftheMscMJchannelsbothbyCPZaswellasbyTNP
MscMJ(Fig.4,B–D).Inasymmetricbuffercontaining200 occurred within tens of minutes, comparable to the activa-
mMKClplus5mMMgCl thechannelhadaconductance tion of MscS by the two amphipaths. This result suggested
2
of g 5 270 6 19 pS (n 5 5). When the bath solution was asimilarmechanismofactivationforboththebacterialand
exchanged to 600 mM KCl plus 5 mM MgCl the reversal archaeal MS channel via preferential insertion of CPZ and
2
potentialE shifted;18mVtowardthereversalpotential TNP into one of the two monolayers of the lipid bilayer of
rev
for potassium. From six different experiments we obtained the spheroplast membrane.
E 5 18 6 2 mV. Using this value we calculated the
rev
permeability ratio of MscMJ for potassium versus chloride
ofP /P ’6,whichindicatedapreferenceofthechannel Effect of expression of MscMJ on E. coli growth
K Cl
forcationsoveranions(Fig.4B).Thus,MscMJselectivity
BecauseithasbeenshownthatMscLandMscSfunctionas
differs from the one of MscS, which was shown to have a
safety valves in osmotically challenged bacteria (Levina et
slight preference for anions over cations with P /P ’ 3
Cl K al., 1999) we examined the effect of expression of MscMJ
(Martinacetal.,1987).Whenthechannelselectivityamong
on the growth of E. coli cells (Fig. 6). E. coli growth was
mono- and divalent cations was examined the selectivity
significantly reduced when expression of mscMJ was in-
sequence of MscMJ for monovalents corresponded to the
duced with IPTG compared with noninduced cell culture.
Eisenman sequence I (Hille, 1992) suggesting that a weak
The effect of the mscMJ expression was partially reversed
anionic site formed a selectivity filter in the channel pore.
when the cells were grown in media of higher osmolarity
The following preference for monovalent cations was ob-
containingeither300mMNaCl,300mMKCl,or600mM
tainedfromthecurrent-voltageplots:Cs15Rb1.K1. sorbitol compared with standard LB media (p , 0.05,
Na15Li1.FordivalentcationsthesequencewasCa21.
ANOVA).
Ba215Mg2.SelectivityforCs1andRb1wassignificantly
differentfromthechannelselectivityforK1andCa21(p,
0.05, ANOVA). Similarly, selectivity for Ba21 and Mg21 DISCUSSION
differed significantly from the selectivity for Na1 and Li1
(p,0.05).Overall,Cs15Rb1.Ca215K1.Ba215 In search of an archaeal homolog of E. coli MscL we
Mg21.Na15Li1.Interestingly,amongdivalentcations identified and cloned a gene from the archaeon M. jann-
aschii using the TM1 domain of E. coli MscL as a genetic
calcium was the most permeant ion (Fig. 4, C and D, and
probe against the M. jannaschii genome. Furthermore, we
Table 1).
demonstratedthatthenewlyidentifiedarchaealMSchannel
that we named MscMJ shares a common ancestral origin
with two types of prokaryotic MS ion channels, MscL and
Effect of amphipaths on MscMJ
MscS. Phylogenetic evidence presented in this study sug-
Cationic chlorpromazine (CPZ) and anionic trinitrophenol gests that prokaryotic MS channels may have originated
(TNP) were shown to activate reversibly MscS in giant from an mscL-like molecule via gene duplication of the
spheroplastsofE.coli(Martinacetal.,1990).Therefore,we ancestralprogenitorgenefollowedbydivergence.Thisim-
examinedtheeffectofthetwoamphipathsonMscMJ(Fig. plies that bacterial MS channels are most likely the evolu-
5, A and B) (note that MscMJ refers only to small conduc- tionary predecessors of the archaeal MS channels in accor-
tanceevents;activationoflargerconductanceeventsbythe dancewiththepresentorganizationofthephylogenetictree
amphipaths was never observed in this study). The activa- (Pace, 1997).
BiophysicalJournal80(1)229–240
MechanosensitiveChannelofM.jannaschii 237
FIGURE 5 Activation of MscMJ by amphipaths.
MscMJ channels were activated by amphipaths in
inside-out excised patches of E. coli giant sphero-
plasts.The20mMchlorpromazine(CPZ;A)and500
mM trinitrophenol (TNP; B) reversibly activated the
channels.Recordingswereobtainedat10and20min
aftertheamphipathswereaddedtothebathsolution.
Pipettevoltagewasheldat250mVand150mVand
pressurewas250mmHgand280mmHgduringthe
CPZandTNPexperiments,respectively.
Evolution of prokaryotic MS channels otic MS channels, which again indicates their common
heritage.
Comparative sequence analysis of known archaeal genes
Several findings in our study support the idea of the
withthegeneticmakeupofbacterialandeukaryoticlinesof
evolutionofprokaryoticMSchannelsviaduplicationofan
descentrevealedthatsomegenes,especiallythoseinvolved
MscL-like molecule. Sequence alignments used in this
in replication, transcription, and translation, resemble eu-
study indicated that two MscL-like TM1 structural motifs
karyoticgenes.However,geneswhosefunctionisrelatedto
have been preserved during evolution within the sequence
energyproduction,celldivision,andmetabolismaresimilar
ofMscMJ,i.e.,MJ0170(Fig.1A),whichfurtherrevealeda
tothosefoundinbacteria.Furthermore,M.jannaschiigenes
remarkablesimilarityandphylogeneticrelationshipwithits
that control the transport of inorganic ions such as potas-
archaeal and bacterial homologs (Fig. 2, A and B). Such
sium and sodium across the cell membrane are very bacte-
ria-like, indicating that the ion transport pathway was de- multiplerepetitionoftheprimarystructuraldomainsofthe
rived from a common ancestor (Morell, 1996). In addition, protein points to a duplication of the mscL-like progenitor
MS channels have been implicated to play a role in the genefollowedbydivergencesimilartothatproposedforthe
regulationofturgorpressure,whichisessentialfordivision evolution of voltage-gated ion channels (Strong et al.,
and growth of bacterial cells (Csonka and Epstein, 1996). 1993). Significantly, the conserved residues include those
Consequently,itwouldbeexpectedthatarchaealMSchan- that were identified to be important for the MscL function.
nelsaremorecloselyrelatedtobacterialratherthaneukary- Whenmutated,severalofthoseresidues,suchasG22(G23
BiophysicalJournal80(1)229–240
238 KlodaandMartinac
MscMJ shares with MscL and MscS is largely matched by
the similarity in functional properties of these prokaryotic
MS channels.
Mechanosensitivity of MscMJ
In terms of its mechanosensitivity MscMJ is functionally
more closely related to MscS than MscL, as MscS
(i.e.,YggB) and MscMJ require less pressure for activation
than does MscL (Berrier et al., 1996). It can be shown that
bymultiplyingp andaonecanobtainanestimateofthe
1/2
free energy of activation for a MS channel (see Materials
and Methods):
G 5p a5DG /kT (1)
MGC 1/2 o
Consequently, for MscMJ DG ’ 5kT, which is approxi-
o
mately three times less than DG 5 18.6kT estimated for
o
FIGURE 6 TheeffectofexpressionofMscMJonE.coligrowth.E.coli MscL (Sukharev et al., 1999) but is similar to DGo 5 7kT
cells expressing mscMJ were growth inhibited. mscMJ gene expression obtained for MscS (Martinac, unpublished; see Materials
wasinducedwithIPTGoncethecellculturereachedmid-logphaseOD600 and Methods). It was previously shown that the negative
;0.6, which corresponded to 2–2.5 h before addition of IPTG (marked
pressure necessary to activate MscS is about two times
with the arrow). The growth was partially rescued in media of high
smallerthanthepressurerequiredforactivationofMscLin
osmolarity containing either 300 mM NaCl, 300 mM KCl, or 600 mM
sorbitol.Fandf,growthcurvesofnoninducedandinduced-with-IPTGE. giant spheroplasts of E. coli (Blount et al., 1996; Berrier et
colicellsharboringpQE-32MJ0170plasmid,respectively.Opensymbols al., 1996). Like MscS, MscMJ could also be activated by
representgrowthcurvesafterinductionwithIPTGinmediacontainingthe voltage,furtherindicatingaclosefunctionalrelationshipto
followingosmoprotectants:KCl(E),NaCl(M),andsorbitol((cid:130)).
MscS (Martinac et al., 1987).
Lipid bilayers of cell membranes of Archaea consist of
of MscMJ), G26 (G27 of MscMJ), and K31 (K32 of diphytanylglycerol-diether or -tetraether or both (Doolittle,
MscMJ) (Fig. 1 A), produce bacterial phenotypes with im- 1999) and are therefore chemically very different from
pairedgrowthandincreasedMscLsensitivitytopressurein phospholipids of bacteria or eukaryotes, which are charac-
patch-clamp experiments (Blount et al., 1997; Ou et al., terized by ester linkages between glycerol and acyl chains
1998). According to the three-dimensional crystal structure (Kates, 1993). However, in terms of physical properties
ofMscL(Changetal.,1998)theseresiduesarelocatedator relevant to gating of MS channels by mechanical force,
nearthechannelgateandthusseemtobeimportantforthe lipidsofM.jannaschiiseemnottodifferfromphospholip-
channel gating. ids,becauseMscMJexhibitsmechanosensitivityinbilayers
The lack of MscL homologs in the current archaeal of azolectin liposomes.
databasesandthepresenceoftheMscL-likestructuralmotif
amongMscMJhomologsrepresentingthreespeciesbelong-
ing to two different phyla further suggest the origin of Conductance and selectivity of MscMJ
prokaryotic MS channels from a common ancestor, which Using the value for the channel conductance g 5 270 pS
mostlikelyresembledMscL(Fig.2,AandB).Thecommon
(Fig.4,BandC)weestimatedthesizeofthechannelpore
ancestryandevolutionarydivergenceisalsoreflectedinthe to be d ’ 9 Å. The following expression was derived
phylogenetictreeofalignedsequences,whichareclustered pore
fromHille’smodel(Hille,1968)tocalculatethediameterof
into three main branches.
the channel pore:
MscL(136residues)isapproximatelytwoandhalftimes S ˛S D D
smallerthanMscS(286residues)oritsarchaealcounterpart rg p p 2 4pl
MscMJ(350residues).Themostdivergedhomologs,which dpore5 p 2 1 2 1 rg , (2)
occupy the third branch on the phylogenetic tree, are ap-
proximately three times larger than MscS or MscMJ ho- where the resistivity of the recording solution rwas mea-
mologs.Suchgradualincreaseinthesizeofrelatedproteins sured as 49.7 Vcm and l ’ 40 Å was the bilayer thickness
harboringthebasicpattern,whichcanbetracedbacktothe (Cruickshank et al., 1997). Thus, the pore of MscMJ is
MscL sequence, further supports the common ancestry, approximately three times smaller than that of the MscL
gene duplication and divergence. pore,whichwasestimatedtobe;35Å(Cruickshanketal.,
Asdiscussedfurther,thecommonancestraloriginandthe 1997),andtwotimessmallerthanthesizeoftheMscSpore,
high level of conservation of the primary structure that which from its conductance of ;1 nS (Sukharev et al.,
BiophysicalJournal80(1)229–240