Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane ...

18 Pages·2013·2.38 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.288,NO.16,pp.11294–11303,April19,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Structural Sensitivity of a Prokaryotic Pentameric
Ligand-gated Ion Channel to Its Membrane Environment*□S
Receivedforpublication,January30,2013,andinrevisedform,February28,2013Published,JBCPapersinPress,March5,2013,DOI10.1074/jbc.M113.458133
JonathanM.Labriola‡,AkashPandhare§,MichaelaJansen¶,MichaelP.Blanton§,Pierre-JeanCorringer(cid:1),
andJohnE.Baenziger‡1
Fromthe‡DepartmentofBiochemistry,Microbiology,andImmunology,UniversityofOttawa,Ottawa,OntarioK1H8M5,Canada,
the§DepartmentofPharmacologyandNeuroscienceandtheCenterforMembraneProteinResearch,SchoolofMedicine,Texas
TechUniversityHealthSciencesCenter,Lubbock,Texas79430,the¶DepartmentofCellPhysiologyandMolecularBiophysicsand
theCenterforMembraneProteinResearch,SchoolofMedicine,TexasTechUniversityHealthSciencesCenter,Lubbock,Texas
79430,andthe(cid:1)G5GroupofChannelReceptors,CNRSURA2182,PasteurInstitute,F75015,Paris,France
Background: The lipid sensitivity of the prokaryotic pentameric ligand-gated ion channel (pLGIC), GLIC, is poorly
characterized.
Results:GLICismorethermallystableanddoesnotexhibitthesamepropensitytoadoptanuncoupledconformationasthe
TorpedonAChR.
Conclusion:GLICislesssensitivetoitssurroundingmembraneenvironment.
Significance:TheGLICandnAChRstructuressuggestmolecularfeaturesgoverningthelipidsensitivityofpLGICs.
Althoughtheactivityofthenicotinicacetylcholinereceptor brainfunction,areimplicatedinavarietyofneurologicaldis-
(nAChR)isexquisitelysensitivetoitsmembraneenvironment, orders,andarethetargetsofbothendogenousandexogenous
theunderlyingmechanismsremainpoorlydefined.Thehomol- modulators(1–5).
ogousprokaryoticpentamericligand-gatedionchannel,Gloe- Earlyattemptsatpurifyingandreconstitutingtheprototypic
bacterligand-gatedionchannel(GLIC),representsanexcellent Cys-loopreceptor,theTorpedonicotinicacetylcholinerecep-
model for probing the molecular basis of nAChR sensitivity tor(nAChR),2inlipidbilayersrevealedtheimportanceoflipids
becauseofitshighstructuralhomology,relativeeaseofexpres- inCys-loopreceptorfunction(6).Inthepresenceofbothcho-
sion,andamenabilitytocrystallographicanalysis.Weshowhere lesterol and anionic lipids, the nAChR is stabilized predomi-
thatmembrane-reconstitutedGLICexhibitsstructuralandbio- nantly in an activatable resting conformation (7–10). In their
physicalpropertiessimilartothoseofthemembrane-reconsti- absence, the nAChR adopts an “uncoupled” conformation,
tuted nAChR, although GLIC is substantially more thermally whereagonistbindingfailstoelicitchannelgating(11).Under-
stable.GLIC,however,doesnotpossessthesameexquisitelipid standing the mechanisms by which lipids influence the cou-
sensitivity.Inparticular,GLICdoesnotexhibitthesamepro- plingofbindingandgatingremainscentraltounderstanding
pensity to adopt an uncoupled conformation where agonist lipid-nAChR interactions. Uncoupled nAChRs may also have
bindingisuncoupledfromchannelgating.Structuralcompari- broader significance in that neuronal nAChRs that are func-
sonsprovideinsightintothechemicalfeaturesthatmaypredis- tionallyuncoupledhavebeenobservedinheterologousexpres-
posethenAChRtotheformationofanuncoupledstate. sionsystemsandmayplayaroleinthephysiologicalresponse
tonicotine(12).Unfortunately,detailedinsightintothemech-
anismsoflipid-dependentuncouplingremainselusive(11,13).
Cys-loopreceptorsmediaterapidchemicalcommunication A major bottleneck is the inability to reliably express large
between cells in the nervous system by opening a transmem- quantities of Cys-loop receptors with site-directed mutations
braneionchannelacrossthesynapticmembraneinresponseto designedtotestpotentialmodelsoflipid-nAChRinteractions.
neurotransmitterbinding.Channelopeningleadstotheflowof Severalprokaryoticpentamericligand-gatedionchannels
either cations or anions down their electrochemical gradient (pLGICs)homologoustotheeukaryoticCys-loopreceptors
intothecell,resultingineitheranexcitatoryoraninhibitory haverecentlybeenidentified,andcrystalstructuresoftwoof
response,respectively.Cys-loopreceptorsplayacentralrolein thesesolvedathighresolution(14–17).ProkaryoticpLGICs
provideanopportunityforprobingthemechanismsofCys-
*ThisworkwassupportedbygrantsfromtheCanadianInstituteofHealth loopreceptor-lipidinteractionsatamolecularlevelbecause
ResearchandtheNaturalSciencesandEngineeringResearchCouncilof oftheirhighstructuralhomology(Fig.1),theirrelativeease
Canada(toJ.E.B.),byaBoursesd’ÉtudespourlaFrance(toJ.E.B.)from
l’AmbassadedeFranceauCanada,bygrantsfromtheSouthPlainsFoun-
dationandtheCenterforMembraneProteinResearch,TexasTechUniver- 2The abbreviations used are: nAChR, nicotinic acetylcholine receptor;
sityHealthScienceCenter(toM.P.B.andM.J.),andbyagrantfromthe GLIC,Gloebacterligand-gatedionchannel;aso-GLIC,GLICinasolectin
NationalInstitutesofHealthGrantNS059841(toM.J.). membranes;aso-nAChR,nAChRinasolectinmembranes;Carb,carba-
□S ThisarticlecontainssupplementalFigs.S1–S6. mylchoilne; DDM, dodecylmaltoside; EcoLip-GLIC, GLIC in E.coli lipid
1To whom correspondence should be addressed: Dept. of Biochemistry, membranes,pLGIC,pentamericligand-gatedionchannel;PC,1-palmotyl-
Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd., 2-oleoyl-sn-glycero-3-phosphocholine;PC-GLIC,GLICinPCmembranes;
Ottawa,ONK1H8M5,Canada.Tel.:[phone],ext.8222;Fax:613-562- PC-nAChR, nAChR in PC membranes; PA, phosphatidic acid; Chol,
5440;E-mail:[email protected]. cholesterol.
11294 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER16•APRIL19,2013
This is an Open Access article under the CC BY license.
LipidSensitivityofaProkaryoticpLGIC
GLIC Expression and Purification—GLIC was expressed in
theC43strainofE.colitransformedwithapET-20b((cid:1))vector
containingtheDNAsequenceforthemaltose-bindingprotein
fusedtotheNterminusofGLICthroughathrombin-sensitive
peptidelinker(14).Briefly,2-literculturesofthetransformed
C43 cells were grown in 2YT medium containing 50 (cid:2)g/ml
ampicillin at 37°C to an A of (cid:2)1.2, and the cultures were
600
inducedovernightat30°Cwith100(cid:2)Misopropyl(cid:3)-D-thioga-
lactopyranoside. The cells were harvested, resuspended in
BufferA(250mMNaCl,25mMTris,pH7.2)inthepresenceof
Roche CompleteTM antiprotease tablets, and lysed with an
AvestinEmulsiflex-C5homogenizer(Ottawa,Canada).Mem-
branefractionsweresolubilizedin1%DDMinBufferA,and
theGLICfusionproteinwasboundtoanamyloseaffinityresin.
Aftertreatmentwithplasminogen-freethrombinfromCalbi-
ochem(Gibbstown,NJ),GLICwaselutedin0.02%DDMand
FIGURE1.ThestructuresofGLIC(A,ProteinDataBankcode3EAM)and
theTorpedonAChR(B,ProteinDataBankcode2BG9).Bothstructuresare furtherpurifiedonaSuperpose610/300gelfiltrationcolumn
sideviewsfromwithintheplaneofthemembrane.Coloringhighlightsthe (GEHealthcare).ThehighlypurifiedGLICwaspassedquickly
domainstructure:theextracellularagonist-bindingdomain(ABD)inred,the
through1mlofamyloseresintoremovesmallamountsofan
transmembranedomain(TMD)inblue,andthecytoplasmicdomain(CD)in
green. endogenousamylosebindingproteinthatco-eluteswithGLIC
fromthesizeexclusioncolumn.
Membrane Reconstitution of GLIC—The purified GLIC in
of expression in bacterial systems, and their amenability to
0.02%DDMinbufferAwasslowlydilutedatleast1:4withlipids
crystallographicanalysis.Althougharecentstudysuggested
solubilizedin0.625%cholateinBufferAtogivethedesiredfinal
thatcholesteroldepletionmayinfluencethegatingkinetics
lipidtoproteinratio,inthiscase2:1(w/w)(19,20).Aftergently
of one pLGIC, GLIC (18), the broader effects of lipids on mixing for (cid:2)30 min, the protein/detergent/lipid mixture was
prokaryotic pLGIC structure and function remain to be
dialyzedfivetimesat4°Cagainst2litersofBufferAleadingto
studied. In fact, a reliable protocol for reconstituting pro-
aturbidsolutionofproteoliposomes,whichwereharvestedby
karyoticpLGICsinmembranesatlipidtoproteinratiosthat ultracentrifugationat100,000(cid:3)gfor2h.Thepelletedmem-
are amenable to spectroscopic studies, particularly in the
branes were resuspended and layered onto a discontinuous
context of probing the electrophysiologically silent uncou- sucrosedensitygradient.Afterultracentrifugationat100,000(cid:3)
pledstate,hasyettobedeveloped.
gfor20hinaBeckmanSW41swingingbucketrotor,sequential
Here,wecomparethestructuralandbiophysicalproperties 400-(cid:2)l aliquots were removed and assayed for both protein
ofmembrane-reconstitutedGLICwiththoseofitshighlylipid- (BCATM assay from Thermo-Pierce) and lipid (phospholipid
sensitiveeukaryotichomolog,theTorpedonAChR.Thesecom-
C/choline assay from Wako Chemicals (Richmond, VA)).
parisons were performed both in membranes that support
Appropriatefractionswerepooledandeitherdialyzedafurther
GLIC/nAChRfunctionandinthosethatpromoteformationof
five times or subjected to several centrifugation resuspension
the uncoupled nAChR. Our results suggest that although the
cyclestoremovesucrose.
gatingpropertiesofGLICaresensitivetoitslipidenvironment,
Reconstituted nAChR Membranes—nAChR purification
GLIC does not possess the exquisite lipid sensitivity of its
andreconstitutionwasperformedasdescribed(11,21).The
eukaryotic homolog. In particular, GLIC does not exhibit the
nAChR was solubilized from Torpedo electroplaques with
same propensity to adopt an uncoupled conformation where
1% sodium cholate and purified on a bromoacetylcholine
agonist binding is uncoupled from channel gating. Structural
affinity column in the presence of lipid. Protein was eluted
comparisons provide insight into the chemical features that
with10mMCarb,andthenthemembranewasreconstituted
may predispose Cys-loop receptors to the formation of an
bydialysis.
uncoupledstate.
FourierTransformInfraredSpectroscopy—Hydrogen/deute-
riuminfraredspectra(Fig.2A)wererecordedonaDigilab(now
EXPERIMENTALPROCEDURES
AgilentTechnologies(SantaClara,CA))FTS40spectrometer
Materials—Soybean asolectin (L-(cid:1)-phosphatidylcholine, using a Golden-GateTM attenuated total internal reflection
typeII-S),sodiumcholate,carbamylcholine(Carb),andaman- accessory(SpecAc;Oprington(Kent,UK)).10(cid:2)lofthemem-
tadine were from Sigma. Escherichia coli polar lipid extracts brane-reconstituted GLIC ((cid:2)1 mg/ml) in 2 mM phosphate
and 1-palmotyl-2-oleoyl-sn-glycero-3-phosphocholine were buffer (pH 7.0) were dried down onto the accessory under a
fromAvanti(Alabaster,AL).Torpedocalifornicaelectroplaque gentlestreamofN gas.Aftercollectinga64-scanspectrumat
2
organwasfromAquaticResearchConsultants(SanPedro,CA). 4cm(cid:4)1resolution,10(cid:2)lofbufferAin2H Owasadded,and
2
Dodecylmaltoside (DDM) was from Affymetrix (Santa Clara, another64-scanspectrumwascollected.
CA). [125I](cid:1)-Bungarotoxin (107 Ci/mmol) was obtained from ForthemoredetailedamideIbandanalyses(Fig.2B)and
PerkinElmer Life Sciences. Nonradioactive (cid:1)-bungarotoxin for thermal stability measurements, the membrane-recon-
wasfromBiotium,Inc.(Hayward,CA). stituted GLIC was exchanged into 2 mM phosphate 2H O
2
APRIL19,2013•VOLUME288•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 11295
LipidSensitivityofaProkaryoticpLGIC
enhancement was performed between 1900 and 1300 cm(cid:4)1
using a (cid:4)(cid:5) 7 and a Bessel smoothing function set to 70%.
Thermaldenaturationwasmeasuredasthechangeinintensity
at 1681 cm(cid:4)1 and was evaluated using GraphPad Prism (La
Jolla,CA)(11).
Electrophysiology—Electrophysiologywasperformedusinga
two-electrode voltage clamp apparatus (OC-725C oocyte
clampfromWarnerInstruments(Hamden,CT)orasdescribed
elsewhere(23).Exceptwherenoted,theoocyteswereinjected
with100ngofmembrane-reconstitutedGLICornAChRor6.6
ngofGLICmRNAandallowedtoincubatefor1–2daysat16°C
inND96(cid:1)buffer(5mMHEPES,96mMNaCl,2mMKCl,1mM
MgCl ,1mMCaCl ,and2mMpyruvate).Injectedoocyteswere
2 2
placedinaRC-1Zoocytechamber(HarvardApparatus,Ham-
den,CT)containingMESbuffer(140mMNaCl,2.8mMKCl,2
mM MgCl , and 10 mM MES, pH 7.0). Currents through the
2
plasma membrane in response to pH jumps were measured
withthetransmembranevoltage-clampedatbetween(cid:4)20and
(cid:4)60mV.TheoocytechamberwasperfusedwithMESbufferat
arateof(cid:2)5ml/min.
(cid:1)-Bungarotoxin Binding Assays—Individual oocytes were
incubated with 2.58 nM [125I](cid:1)-bungarotoxin for 2 h at room
temperature.Theoocyteswerewashedthreetimeswith1mlof
ice-coldbuffercontaining1%BSA,andthenbindingwasquan-
tifiedby(cid:4)-counting.Nonspecificbindingwasdeterminedwith
oocytesfromthesamebatchincubatedwith5(cid:2)Mnonlabeled
(cid:1)-bungarotoxin.
RESULTS
MembraneReconstitutionofGLIC—GLICwasreconstituted
intomembranesatalipidtoproteinratioof2:1(w/w),because
thisratioisidealforspectroscopicstudies,suchasthoseused
FIGURE2.Structuralcomparisonsofmembrane-reconstitutedGLICand
thenAChRasprobedbyinfraredspectroscopy.A,infraredspectraofaso- previouslytocharacterizetheuncouplednAChR(see“Discus-
GLIC recorded after gentle drying from 1H2O buffer (solid black line) and sion”)(11).Toreconstituteintomodelmembranes,DDM-sol-
immediatelyafteradditionof2H O(dashedgrayline).Notetheimmediate
changes in amide I band shape2 (1700–1600 cm(cid:4)1) and the immediate ubilized GLIC (supplemental Fig. S1) was diluted below the
decreaseinamideIIbandintensity(1547cm(cid:4)1),bothindicativeoftherapid criticalmicellarconcentrationforDDMwithacholate-solubi-
peptideN-1H/N-2Hexchangeofsolvent-exposedpeptidehydrogens.Similar
lized lipid solution, and the protein/detergent/lipid mixture
spectraleffectsareobservedforotherCys-loopreceptors(supplementalFig.
S2).B,infraredspectrarecordedafter72hofequilibrationin2H Oat4°Cfrom wasdialyzedextensivelytoremoveresidualDDMandcholate.
2
aso-GLIC(spectrumi),EcoLip-GLIC(spectrumii),PC-GLIC(spectrumiii),aso- SucrosedensitygradientsshowedthatGLICefficientlyincor-
nAChR(spectrumiv),andPC-nAChR(spectrumv).Theleftcolumnshowsthe
porates into model membranes. Neither residual DDM nor
secondarystructure-sensitiveamideIbandbothbefore(graytraces)andafter
resolutionenhancement(blacktraces)(intensityscalingarbitrary).Theright cholate could be detected in the reconstituted membranes
columnshowstheamideIIbandineachspectrum.Therelativeintensityof
usinganinfraredbasedassayfordetergent(24).GLICwasini-
theamideIIvibrationisbestassessedrelativetotheintensityoftheadjacent
broadpeakbetween1560and1600cm(cid:4)1,becauseofasparticandglutamic tiallyreconstitutedintomembranescomposedofeitherasolec-
acidresidues.Allofthepresentedspectraaretheaveragesofseveralspectra tin lipids (aso-GLIC) or E.coli polar lipid extracts (EcoLip-
recordedfromatleasttwodifferentpurification/reconstitutions.
GLIC).Thesemembraneswerechosenbecauseasolectinlipids
(aswellasdefinedmembranescomposedofphosphatidylcho-
buffer, pH 7.0, for precisely 72 h at 4°C and then stored at line, phosphatidic acid, and cholesterol (PC/PA/Chol) 3:1:1
(cid:4)80°Cuntiluse.Approximately125(cid:2)gofGLICwasdepos- (molarratios))stabilizethenAChRinafunctionalrestingcon-
ited on a CaF window with a gentle stream of N gas fol- formationandminimizeGLICaggregation(18),whereasE.coli
2 2
lowedbyrehydrationwith8(cid:2)lofBufferAin2H O,pH7.0. polar lipid extracts likely resemble the natural lipid environ-
2
A 4000-scan spectrum was collected at 2 cm(cid:4)1 resolution ment of GLIC. We expected both membranes to stabilize an
and 22.5°C on a Digilab (now Agilent Technologies) activatable“resting”conformation.
FTS7000spectrometer.128-scanspectrawerethenacquired StructureofMembrane-reconstitutedGLIC—Infraredspec-
at1°Cintervalsfrom35to90°Cwitha20-minequilibration traofbothaso-GLICandEcoLip-GLICgentlydriedfrom1H O
2
periodbetweeneachtemperaturejump. buffer(toeliminatetheintenseoverlappingvibrationsofbulk
The spectra were analyzed using GRAMS/AI software water)exhibittworelativelyintenseproteinbands:theamideI
(ThermoScientific,Waltham,MA).Residualwatervaporwas bandbetween1700and1600cm(cid:4)1andtheamideIIbandcen-
subtracted using the method of Reid et al. (22). Resolution terednear1547cm(cid:4)1(Fig.2A).TheamideIbandisduepri-
11296 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER16•APRIL19,2013
LipidSensitivityofaProkaryoticpLGIC
marilytopeptidebackboneC(cid:5)Ostretchingvibrationsandis and/or loop/random structures, is still observed in spectra
sensitive to protein secondary structure. The amide II band, recordedafterprolongedexposureofGLICto2H O.Thissug-
2
primarily due to peptide backbone C-N stretching vibrations geststheexistenceofalargepopulationofexchange-resistant
coupledtoN-1Hbendingvibrations,isalsosensitivetosecond- andlikelytransmembrane(cid:1)-helices,ashasbeencharacterized
arystructurebutisusedprimarilytomonitortheexchangeof forthenAChR(25,28).Alargepopulationofsolvent-shielded
peptideN-1HtoN-2H.TheamideIIbandshiftsdowninfre- peptide hydrogens is also suggested by the residual amide II
quencyfrom1547cm(cid:4)1tonear1450cm(cid:4)1in2H O,thedown- bandintensity(Fig.2B).Theproportionofexchange-resistant
2
shiftedvibrationoftenreferredtoastheamideII(cid:6)band. peptide hydrogens in both aso-GLIC and EcoLip-GLIC is
The GLIC amide I contour observed in spectra dried from slightly higher than in aso-nAChR, although aso-GLIC
1H O exhibits a peak maximum near 1655 cm(cid:4)1, due to the exchangestoagreaterextentthanEcoLip-GLIC(see“Discus-
2
overlappingvibrationsof(cid:1)-helixandloop/randomsecondary sion”). The proportion of exchange-resistant peptide hydro-
structures,andaprominentbroadshoulderbetween1640and gens in aso-nAChR was estimated to be (cid:2)40%, sufficient to
1625 cm(cid:4)1, primarily due to the vibrations of (cid:3)-sheet. The account for the peptide hydrogens located in the transmem-
amide I band shape is similar to that observed for functional brane domain (11, 29). The greater proportion of exchange-
membrane-reconstitutedTorpedonAChRs(supplementalFig. resistantpeptidehydrogensinGLICislikelyduetothelackof
S2), suggesting a similar mixed (cid:1)-helix/(cid:3)-sheet secondary an intracellular domain: the exchange-resistant transmem-
structure. The slightly increased intensity between 1640 and brane domain of GLIC makes up a greater proportion of the
1620cm(cid:4)1indicatesaslightlyhigher(cid:3)-sheetcontentinGLIC. totalproteinstructure(Fig.1).
Themixed(cid:1)-helix/(cid:3)-sheetsecondarystructureoftherecon- TheonenotabledifferencebetweenthedeconvolvedamideI
stituted GLIC was confirmed by recording spectra in 2H O contoursofGLICandthenAChRistheexistenceinthelatterof
2
buffer,wherethereisnooverlapofthesolventvibrationswith amoreintensebandcenterednear1645cm(cid:4)1causedbydown-
theamideIband(Fig.2B).TheresultingamideIcontourstill shiftedandthuspeptideN-1HtoN-2Hexchanged(cid:1)-helicaland
exhibits features at frequencies characteristic of both (cid:1)-helix loop/randomstructures.Thisbandappearsimmediatelyafter
(1655cm(cid:4)1)and(cid:3)-sheet(1640–1625cm(cid:4)1),asshownclearly exposureofthenAChRto2H O,suggestingthattheaffected
2
by resolution enhancement. Curve fitting (supplemental Fig. structuresaresolvent-exposed(28).Solvent-exposed(cid:1)-helical
S3)suggestsroughly35%(cid:1)-helixand40%(cid:3)-sheet(supplemen- andloop/randomstructuresexistintheintracellulardomainof
talFig.S3),estimatesthatcomparewellwiththecrystalstruc- thenAChR.Thelackofasimilarlyintensebandcenterednear
ture(roughly35%(cid:1)-heliceswith(cid:7)5residuesand30%(cid:3)-sheet). 1645cm(cid:4)1intheGLICspectraisconsistentwiththeabsenceof
Theslightoverestimateof(cid:3)-sheetcontentcouldreflectdiffi- anintracellulardomaininGLIC.
culties precisely defining the start and end point of each Finally,thethermalstabilitiesofbothaso-GLICandEco-
(cid:3)-strand in the crystal structure. In addition, spectra of the Lip-GLICwereexaminedbyrecordinginfraredspectraasa
(cid:1)-helicalproteinmyoglobin(no(cid:3)-sheet)exhibitweakintensity functionoftemperature.Inbothcases,increasingtempera-
between 1640 and 1620 cm(cid:4)1, demonstrating that structures ture led to changes in amide I band shape characteristic of
otherthan(cid:3)-sheetcancontributeweakintensitytothisregion protein denaturation (supplemental Fig. S4), confirming
(25). that the reconstituted GLIC adopts a folded structure. It is
Notethatwerecordedspectrabothimmediatelyafterexpo- noteworthythatthepreviously“exchange-resistantpeptide
sureto(Fig.2A)andafterprolongedequilibrationwith2H O hydrogens” in the transmembrane domain of GLIC under-
2
buffer(72h,4°C)(Fig.2B).Thespectrarecordedimmediately goes complete N-1H to N-2H exchange concomitant with
afterexposureexhibitchangesinbandshapethatresultfrom unfolding. This behavior is similar to that observed for the
thedownshiftsinfrequencyofsolvent-exposedregionsofthe nAChR but differs from that of other membrane proteins,
peptide backbone. Subtle downshifts in the frequencies of such as LacY, which undergoes complete N-1H to N-2H
amide I component bands caused by turn (1700–1670 cm(cid:4)1 exchange at elevated temperatures, but prior to unfolding
region; see supplemental materials), loop/random and/or (20). The latter property of LacY has been attributed to a
(cid:1)-helical structures (1655 cm(cid:4)1 down to (cid:2)1645 cm(cid:4)1), and temperature-induced increase in the conformational
(cid:3)-sheet(primarily1640–1620cm(cid:4)1region)aredetected(26). dynamicsofthetransmembranedomain,whereasthetrans-
TheserapidamideIbandshiftsareaccompaniedbyadropin membranedomainofGLICmaybeconformationally“static”
amide II band intensity, further demonstrating the rapid in the absence of bound agonist. The transmembrane
exchangeofpeptideN-1HtoN-2H.Thespectralchangesareall domainofGLICisalsonotexpectedtoundergosuchlarge
similartothoseobservedimmediatelyuponexposureofother conformationaltransitions(16,17,30).Bothaso-GLICand
Cys-loop receptors to 2H O (supplemental Fig. S2) (27) but EcoLip-GLIC have higher thermal stabilities of 68.8 (cid:8) 1.5
2
differfromthosetypicallyobservedforproteinswithdifferent and 73.7 (cid:8) 1.4°C, respectively, than that of the nAChR
tertiaryfolds(26).Theresultsemphasizetheexistenceofasub- (cid:2)55°C(Fig.3andTable1).Thehigherthermalstabilityof
stantial population of solvent-exposed secondary structures, GLICisconsistentwithitsenhancedsuitabilityforcrystal-
consistent with the large solvent-exposed extramembranous lization(31).
agonist-bindingdomaininGLIC(16,17). GatingPropertiesoftheMembrane-reconstitutedGLIC—Al-
OnenotablefeatureoftheGLICspectraisthattherelatively thoughreconstitutedmembranesatalipidtoproteinratioof
intense1655cm(cid:4)1amideIcomponentband,whichisdueto 2:1 (w/w) are ideal for spectroscopic studies, they are not
thevibrationsofprotiatedandthussolvent-shielded(cid:1)-helical useful for direct measurements of channel activity using
APRIL19,2013•VOLUME288•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 11297
LipidSensitivityofaProkaryoticpLGIC
FIGURE 4. The Carb-induced response of membrane-reconstituted
nAChRafterinjectionintoandconsequentfusionwiththeplasmamem-
braneofXenopusoocytes.A,currentsinducedby500(cid:2)MCarbweremea-
FIGURE3.RepresentativethermaldenaturationcurvesforGLIC(black suredat(cid:4)20mVholdingpotentialfromoocytesinjectedwithaso-nAChR
lines)andthenAChR(graylines)indifferentmembranes.Thedenatur-
(lefttrace)andPC-nAChR(righttrace).B,abargraphcomparingmaximal
ationcurvesareforaso-GLIC(Œ),EcoLip-GLIC(●),PC-GLIC((cid:2)),aso-nAChR recordedcurrents,eachnormalizedtothenumberof[125I](cid:1)-bungarotoxin
(‚),PC/PA/Chol-nAChR((cid:3)),andPC-nAChR(f).Eachcurvewasfitwitha (BTX)-bindingsites,inducedby300(cid:2)Macetylcholinefromindividualoocytes
Boltzmann sigmoid from which T and Boltzmann slope were calculated
d microinjectedwith125ngofaffinity-purifiedTorpedonAChRproteinrecon-
usingGraphPadPrismsoftware(Table1)(seesupplementalmaterialsand stitutedineitherPC/PA/Chol(3:1:1molarratio;0.124(cid:8)0.061(cid:2)A/[125I](cid:1)-
Ref. 11). The Boltzmann slope decreases with increasing cooperativity of bungarotoxin-binding sites/oocyte; n (cid:5) 5) or PC lipid vesicles (0.0034 (cid:8)
unfolding. 0.0006(cid:2)A/[125I](cid:1)-bungarotoxin-bindingsites/oocyte;n(cid:5)6).
TABLE1
braneandthusthenAChRretainingits“reconstitutedmem-
ThermalstabilityofGLIC
brane”lipidenvironment.SuchclusteringofthenAChRon
Reconstitution T Boltzmannslope n
d
theoocytesurfacehasbeensuggestedfrompreviousstudies,
aso-GLIC 68.8(cid:8)1.5 2.44(cid:8)0.16 5
EcoLip-GLIC 73.7(cid:8)1.4 1.98(cid:8)0.22 5 (32).PC-nAChRmayalsobelockedinanuncoupledconfor-
PC-GLIC 70.4(cid:8)0.9 2.35(cid:8)0.20 4 mation because the oocyte plasma membrane lipids do not
aso-nAChR 55.4(cid:8)1.0 2.25(cid:8)0.01 2
PC/PA/Chol-nAChRa,b 56.4(cid:8)0.8 2.08(cid:8)0.20 6 promote transitions from uncoupled to coupled conforma-
PC-nAChRb 52.4(cid:8)0.1 3.08(cid:8)0.12 6 tions. Recent studies suggest that the uncoupled nAChR in
aThenAChRinmembranescomposedofPC,phosphatidicacid,andcholesterol. longchainPCmembranescanundergoligand-inducedtran-
bFromdaCostaandBaenziger(11).
sitionstocoupledconformations(datanotshown).Finally,it
electrophysiological approaches. Instead, we indirectly ispossiblethatdetergentsolubilizationleadstoirreversible
probed activity by injecting the reconstituted GLIC mem- effects on nAChR structure and function. The latter seems
branesintoXenopuslaevisoocytesandthenmonitoringthe unlikely,however,giventhatdetergentsolubilizationhasno
appearance of proton-activated currents across the oocyte deleterious effects on the nAChR reconstituted into other
plasma membranes using a two-electrode voltage clamp membranes. In addition, no detectable effects of detergent
apparatus(23,32). solubilizationandPCreconstitutionareobservedonnAChR
Wefirsttestedthefeasibilityofthisapproachusingrecon- secondary structure. Regardless, these control experiments
stituted nAChR membranes of defined function. Carb-in- confirm that the injection of reconstituted nAChR mem-
ducedcurrentsthatdecayslowlybecauseofdesensitization branes into oocytes can, in principle, be used to indirectly
are observed in oocytes injected with either aso-nAChR or assessthegatingactivityofreconstitutednAChRs.
PC/PA/Chol-nAChR,showingthatthefunctionalreconsti- We next characterized the response of uninjected, mock
tutednAChRmembranesfusewiththeoocyteplasmamem- injected (membranes alone), and GLIC mRNA-injected
brane, leading to the appearance of functional nAChRs on oocytes to jumps in proton concentration. Both uninjected
the oocyte surface (16 fmol of binding sites/oocyte, with and mock injected oocytes exhibit weak proton-activated
0.124(cid:8)0.061(cid:2)A/fmol[125I](cid:1)-bungarotoxin-bindingsites/ currentsbecauseofendogenousacid-sensitivechannels(Fig.
oocyte;Fig.4).Incontrast,weakornegligibleCarb-induced 5).Thesecurrents,however,showaroughlylinearresponse
currentswereobservedinoocytesinjectedwiththeuncou- toprotonconcentrationatpHvaluesdownto3.5,areinsen-
pledandnonactivatablePC-nAChR,eventhoughtherecon- sitivetotheGLICporeblockeramantadine,andhadmaxi-
stituted PC membranes fuse with the plasma membrane malcurrentsof0.069(cid:8)0.016(cid:2)A(n(cid:5)4)and0.063(cid:8)0.021
leading to the appearance of nAChR agonist sites on the (cid:2)A(n(cid:5)6),respectively,atthemaximalutilizedprotoncon-
surfaceoftheoocyte(5.5fmolof[125I](cid:1)-bungarotoxin-bind- centration(pH3.5).Incontrast,oocytesinjectedwithGLIC
ing sites/oocyte, with 0.0034 (cid:8) 0.0006 (cid:2)A current/fmol RNA exhibit robust proton-activated cationic currents
binding sites/oocyte; Fig. 4). The lack of activity of the (supplementalFig.S5)withrelativelylargemaximalcurrents
plasma membrane-incorporated PC-nAChR ((cid:2)40-fold re- typically close to 4 (cid:2)amps. A plot of whole cell currents
duction in normalized current levels compared with the versusprotonconcentrationforthelatteryieldedapH for
50
injectedPC/PA/Chol-nAChRs)hasseveralpossibleexplana- activation of 4.82 (cid:8) 0.04 (Fig. 4B), consistent with that
tions.First,thelackofactivitycouldreflectclusteringofthe observedpreviouslyforoocyte-expressedGLIC(14).These
reconstituted membranes within the oocyte plasma mem- proton-activated currents are sensitive to the pore blocker,
11298 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER16•APRIL19,2013
LipidSensitivityofaProkaryoticpLGIC
FIGURE5.Theproton-inducedresponseofmembrane-reconstitutedGLICafterinjectionintoandconsequentfusionwiththeplasmamembraneof
Xenopusoocytes.A,electrophysiologyrecordingsinresponsetopHjumpsfromuninjectedoocytesandoocytesinjectedwithaso-GLIC,EcoLip-GLIC,and
PC-GLIC.Thedose-responsecurvesweremeasuredatarelativelylowmembranepotentialof(cid:4)20mV,becausethisseemedtogeneratemorestablebaselines.
AnelectricalresponsetopH4.0showingtheeffectof150(cid:2)Mamantadine(graybar)isshownontheright(scalingarbitrary).Thelatterwereperformedata
membranepotentialof(cid:4)60mV.B,peakcurrentachieveduponexposureofoocytesinjectedwiththeindicatedreconstitutedmembranesatpH3.5.The
reportedvaluesaretheaverages(cid:8)standarddeviationfromfiverecordingsperformedonfivedifferenceoocytes.C,comparisonofthedose-responsecurves
obtainedfromoocytesinjectedwiththemembrane-reconstitutedGLICorGLICmRNA.Thedose-responsecurvesfromoocytesinjectedwithmembrane-
reconstitutedGLICwerenormalizedassumingapH of2.90accordingtoVelisettyetal.(51).
50
amantadine. 150 (cid:2)M amantadine blocked close to (cid:2)50% of EffectsofanUncouplingLipidEnvironmentonGLICStruc-
thepH-activatedcurrents. tureandFunction—Wenextcomparedthestructuralandfunc-
Oocytesinjectedwitheitheraso-GLICorEcoLip-GLICboth tional properties of aso-GLIC and EcoLip-GLIC with that of
exhibit currents that respond to proton concentration in a GLICreconstitutedintoPCmembranes(PC-GLIC).Themin-
dose-dependentmanner.ThemaximalresponsesatpH3.5of imal PC environment is of interest because it stabilizes the
0.181(cid:8)0.008(cid:2)A(n(cid:5)5)and0.157(cid:8)0.014(cid:2)A(n(cid:5)5),respec- nAChRinanuncoupledconformationthatbindsagonistbut
tively,areroughly3-foldlargerthantheresponsesobservedin doesnotundergoagonist-inducedconformationaltransitions.
mockoruninjectedoocytesyetareconsistentinmagnitudeto AkeygoalofthisworkwastoascertainwhetherGLICexhibits
the Carb-induced currents observed in aso-nAChR-injected thesamepropensitytouncoupleasthenAChRinaPCbilayer.
oocytes.Ofparticularsignificance,theseproton-inducedcur- The uncoupled PC-nAChR exhibits biophysical properties
rentsweresensitivetotheGLICchannelblocker,amantadine, that distinguish it from the predominantly resting state aso-
whereastheendogenousacid-sensitivechannelsinoocytesare nAChR(Figs.2and3).Thesecondarystructure-sensitiveamid
not. These data suggest that the reconstituted GLIC in both IcontoursinspectrarecordedfromPC-nAChRaresimilarto
membranesretainstheabilitytogateopeninresponsetopro- that of aso-nAChR, but deconvolution reveals a downshift in
ton binding. Interestingly, the proton concentration depen- amide I component band intensity from 1655 cm(cid:4)1 to near
dence of GLIC activation is right-shifted relative to the dose 1645cm(cid:4)1.ThischangeinamideIbandshapeoccursasaresult
response observed with mRNA-injected oocytes, suggesting of enhanced levels of peptide N-1H/N-2H exchange in the
weakerprotonsensitivity.Aweakerprotonsensitivityhasalso uncoupledstate,asopposedtoachangeintheglobalsecondary
been observed for GLIC reconstituted into giant unilamellar structure(33).TheenhancedpeptideN-1H/N-2Hexchangeis
asolectinliposomesatthehighlipidtoproteinratiosrequired shownbythereducedresidualamideIIbandintensityinspec-
for electrophysiological studies (18). The right shifted dose trarecordedfromPC-nAChRversusaso-nAChR.
response suggests that GLIC may also clusters within the AmoredetailedanalysisofthepeptideN-1H/N-2Hexchange
oocytesplasmamembrane,thusmaintainingitsreconstituted kinetics of uncoupled versus resting or desensitized nAChRs
lipidenvironment.Theshifteddoseresponsesuggeststhatthe showsthatregionsofthepolypeptidebackbonethatareburied
gating activity of GLIC is sensitive to its membrane from solvent in the resting conformation become exposed to
environment. solventintheuncoupledstate(11).TheuncoupledPC-nAChR
APRIL19,2013•VOLUME288•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 11299
LipidSensitivityofaProkaryoticpLGIC
also undergoes thermal denaturation at temperatures DISCUSSION
slightly lower than aso-nAChR, and the cooperativity of The underlying mechanisms by which lipids modulate
denaturation is reduced (11). These altered biophysical nAChRfunctionhavebeenthesubjectofcontinuousdebatefor
properties of PC-nAChR have been attributed to weakened over 30 years (6). There has long been a consensus that both
interactions leading to an increased physical separation cholesterol and anionic lipids are required in a reconstituted
between the agonist-binding and transmembrane domains membranetoobtainoptimalnAChRfunction(7–10,34–36).It
(11). Weakened interactions could account for the increased hasalsobeensuggestedthatbothlipidsinfluencenAChRfunc-
solventexposure,thereducedcooperativityofunfolding,and tionbyanallostericmechanism,wherebylipidsalterthefunc-
the lack of coupling of binding and gating (supplemental Fig. tionalresponsetoacetylcholinebystabilizingdifferingpropor-
S6). tionsofpre-existingresting(activatable)versusdesensitizedor
Significantly,wedetectlittleifanystructuralorbiophysical uncoupled (inactivatable) conformations (7, 29). The mecha-
differencebetweenPC-GLICandeitheraso-GLICorEcoLip- nisms by which these lipids influence function, however, are
GLIC(Figs.2and3).TheamideIbandshapesofGLICinall not clear. It has been suggested that some act by binding to
three membranes are similar, suggesting a similar global sec- interstitialsitesbetweendifferent(cid:1)-helicesinthetransmem-
ondarystructure(however,seebelow).Thereisnofurthershift branedomain(13,37).Lipidsmayalsointeractdirectlywiththe
inamideIcomponentbandintensityfrom1655cm(cid:4)1downto lipid-proteininterface(11,37–39).Althoughtheconsensusis
near1645cm(cid:4)1afterprolongedexposureofPC-GLICto2H O. thatlipidchemistryandthusspecificlipidsitesarelikelyimpor-
2
There are no changes in the levels of peptide N-1H/N-2H tant,molecularinsightintoanysuchbindingsiteshasyettobe
exchange,asshownbythesimilarresidualamideIIbandinten- obtained.Thelackofmechanisticinsightreflectsboththecom-
sities, although both aso-GLIC and PC-GLIC are more plexityoflipid-nAChRinteractionsandtheinabilitytoexpress
exchangedthanEcoLip-GLIC.Thethermaldenaturationtem- site-directedmutantsofthenAChR,whichcanbeusedtotest
peratureofPC-GLICisalsosimilartothatofbothaso-GLIC proposedmechanismsoflipid-proteininteractions.
andEcoLip-GLIC,andtherearenochangesinthecooperativity ProkaryoticpLGICsprovideanewopportunityforprobing
ofunfolding(Table1). themoleculardetailsofpLGIC-lipidinteractions(14,40).The
In fact, the only substantial difference observed between prokaryoticpLGIC,GLIC,adoptsasimilartertiaryfoldtothat
spectra recorded from PC-GLIC and either aso-GLIC or ofthenAChR.BothGLICandthenAChRarecation-selective
EcoLip-GLIC is a slight broadening of the amide I contour, ionchannels,althoughGLICandthenAChRaregatedbypro-
noticeablenear1630cm(cid:4)1.Thedegreeofthissubtlebroaden- tonsandacetylcholine,respectively.Thestructuralsimilarities
ing, however, varied between PC-GLIC reconstitutions. ofGLICandthenAChR,aswellastheaddedadvantagethat
Althoughthestructuralbasisofthisspectraleffectremainsto GLICcanbeexpressedinsufficientstudiesforbiophysicaland
bedetermined,itcouldreflectvariationsinGLICaggregation crystallographicstudies,haveledtheincreasinguseofGLICas
withinthemembrane.Previousstudieshavesuggestedthatthe a model for probing pLGIC structure (15–17), basic mecha-
monodispersityofthepentamericGLICwithinthemembrane nisms of pLGIC activation (15–17), pLGIC-drug interactions
islipid-sensitive(18).Monodispersitywasfoundtobeidealin (41, 42), etc. The ability to express large quantities of site-di-
asolectinbilayers.GLICmaytendtoaggregatemoreinPCthan rectedmutantsfinallyopensthedoorfordetailedmechanistic
inotherlipidenvironments,leadingtobroadeningoftheamide studiesoflipid-GLICinteractions.Suchstudies,however,first
Iband.Regardless,ourstructuraldatasuggestthatGLICdoes require a detailed characterization of the effects of lipids on
notadoptanuncoupledconformationinPCmembranessimi- GLICstructureandfunction.
lartothatadoptedbyPC-nAChR. Wereporthereasimpleprotocolforreconstitutingthepro-
Finally,weindirectlyassessedwhetherPC-GLICexhibitsthe karyoticpLGIC,GLIC,intodefinedlipidmembranes.Ourpro-
abilitytogateopeninresponsetoprotonbinding(Fig.4).As tocoldiffersfromthestandardvesicledetergentdestabiliza-
noted, very weak or negligible Carb-induced currents are tion approach typically used to form high lipid to protein
observedinoocytesinjectedwithPC-nAChR,eventhoughPC- ratio membranes for electrophysiological studies (18). One
nAChR fuses with the oocyte plasma membrane. In contrast, advantageofourapproachisthehighefficiencyincorpora-
oocytes injected with PC-GLIC exhibit currents that both tion of GLIC into the model membranes (supplementalFig.
respondtoprotonsinadose-dependentmannerandaresensi- S1).Itiseasytoaccuratelyadjustthelipidtoproteinratioof
tivetoamantadineblock.Themaximalcurrentsobtainedupon thereconstitutedmembranes.Inaddition,thisprotocolcan
injection of PC-GLIC are substantially higher than those in be used to form relatively low lipid to protein ratio
mock or uninjected oocytes and are indistinguishable from proteoliposomes.
those observed in oocytes injected with either aso-GLIC or OurgoalwastoreconstituteGLICintomembranesatlipidto
EcoLip-GLIC.ThepH-activatedcurrentsarealsosensitiveto proteinratiosofroughly2:1(w/w)becausetheresultingvesi-
amantadine. The electrophysiological recordings suggest that cles are ideal for structural studies, such as those used previ-
GLICdoesnotadoptaconformationinPCmembranessimilar ouslytocharacterizetheuncouplednAChR.Theselowlipidto
totheuncoupledconformationadoptedbythenAChR.Even proteinratiovesiclescanbeeasilyseparatedfrombothprotein-
theminimalPCmembraneenvironmentappearssufficientto free vesicles and low lipid-protein ratio protein aggregates
stabilizeGLICinaconformationthatgatesopeninresponseto (supplementalFig.S1).Theyareidealforspectroscopicstudies
agonistbinding. becausethescatteringoflightfromthemembranevesicles(per
11300 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER16•APRIL19,2013
LipidSensitivityofaProkaryoticpLGIC
mol of reconstituted protein) is minimized, and the spectral iology (12). A main goal of this work was to assess whether
overlapbetweensignalsfromlipidandproteinislessofanissue. GLIC exhibits a propensity similar to that of the nAChR to
Thehigherdensitylowlipidtoproteinratioproteoliposomes adoptanuncoupledconformationinPCbilayers.
areeasiertopellet(particularlyinhigherdensity2H Obuffers) Although our data show that GLIC activity is sensitive to
2
andthusexchangeinto2H O.Lowlipidtoproteinratiovesicles lipids(seebelow),PC-GLICdoesnotexhibitthestructuralor
2
are also ideal for structural studies using infrared difference functional characteristic of the uncoupled conformation
spectroscopy,atechniquethatdetectsresidue-specificchanges adoptedbyPC-nAChR.Specifically,PC-GLICdoesnotexhibit
in structure of the nAChR upon agonist-binding (27, 43, 44). thesubstantialincreaseinsolventaccessibilityofpolypeptide
ApplicationofthisapproachtoprokaryoticpLGICswouldbe backbone hydrogens, which has been attributed to weakened
particularly informative given the ability to express mutants interactionsbetweentheagonist-bindingandtransmembrane
andthusassigndetectedvibrationalchangestospecificamino domains.PC-GLICdoesnotexhibitthereducedcooperativity
acidsidechains. of thermal denaturation that is observed for PC-nAChR.
Membrane-reconstitutedGLICandthenAChRexhibitsim- Finally, unlike PC-nAChR, which remains inactive even after
ilarstructuralandbiophysicalproperties.Bothproteinsexhibit fusion with oocyte plasma membranes, fusion of PC-GLIC
amixed(cid:1)-helix/(cid:3)-sheetsecondarystructurewithbothasub- leadstoanactiveconformation.Althoughfurtherstudiesare
stantial proportion of the polypeptide backbone exposed to requiredtofullycharacterizethefunctionalpropertiesofGLIC
aqueous solvent and a large proportion of (cid:1)-helical peptides indifferentmembranes,itappearsthatPC-GLICisnotlocked
shieldedfromsolventandlikelyfoundinthetransmembrane inanuncoupledconformationthatisunresponsivetoagonist
channelporedomain.GLICexhibitsahigher(cid:3)-sheetcontent binding.
than the nAChR, as expected given that the predominantly ThefindingthatGLICdoesnotexhibitthesamepropensity
(cid:3)-sheet agonist-binding domain in GLIC makes up a larger as the nAChR to adopt an uncoupled conformation can be
proportionofthetotalproteinstructure(seebelow).Themem- interpretedinlightofourworkingmodelofuncoupling.This
brane-reconstituted GLIC also undergoes thermal denatur- model proposes that lipids influence the coupling of binding
ation, albeit at higher temperatures than the nAChR. These andgatingviatheM4transmembrane(cid:1)-helices(onepersub-
observationsshowthatthereconstitutedGLICmaintainsthe unit)(11),whichareexposedtothesurroundinglipidbilayer
expectedstructuralfold. and thus ideally situated to sense membrane physical and/or
Ontheotherhand,infraredspectroscopydetectsarelatively chemical properties (Fig. 1) (45). Each M4 (cid:1)-helix extends
intense vibration near 1645 cm(cid:4)1 in spectra of the nAChR beyondthebilayertointeractdirectlywiththeCys-loop,akey
because of solvent-exposed (cid:1)-helix and/or loop/random sec- structure at the interface between the agonist-binding and
ondary structures. This vibration could reflect primarily the transmembranedomains.Byinfluencingtheconformationof
solvent-exposed (cid:1)-helix and loop/random secondary struc- the M4 “lipid sensor,” lipids may modulate interactions
turesfoundintheintracellulardomainofthenAChR(Fig.1). betweenM4andtheCys-loop(supplementalFig.S6andRef.
ThelackofsimilarintensityinthisregioninspectraofGLICis 11).WeakenedM4/Cys-loopinteractionscouldleadtoaCys-
consistentwiththelackofanintracellulardomainintheGLIC loop conformational change that ultimately reduces contact
crystalstructure. betweentheagonistbindingandthetransmembranedomains
Weusedanindirectelectrophysiologicalapproachtoprobe andleadstotheuncoupledstate.
theabilityofthereconstitutedGLICtogateopeninresponseto A dynamic equilibrium may exist between coupled and
agonist binding. Injection of membrane-reconstituted GLIC uncoupledconformationswhereM4isboundtightlyortilted
intooocytesledtotheappearanceofprotonactivatedcurrents awayfromtheCys-loop,respectively(46,47).Infact,anequi-
across the oocyte plasma membrane. The oocyte membrane- libriumbetweenM4solvatedbylipidandM4boundtightlyto
incorporated GLIC responds in a dose-dependent manner to theothertransmembrane(cid:1)-helices,M1(cid:1)M3,hasbeendem-
protons,andtheresultingcurrentswereblockedbytheGLIC onstrated for the homologous glycine receptor (48). Signifi-
channelblockeramantadine.Bothourstructuralandelectro- cantly, aromatic-aromatic interactions play a key role driving
physiologicalstudiesthussuggestthatthereconstitutedGLIC associationsbetweenM4andM1(cid:1)M3,thusstabilizingafolded
adoptsanativestructurethatgatesopeninresponsetoagonist structure.InthecontextoftheM4lipidsensormodel,aromatic-
binding. aromaticinteractionscouldplayakeyroledictatingthepropensity
Amajorgoalofthisworkwastocomparethelipidsensitiv- ofM4tobindtightlytoM1(cid:1)M3andtothusformacoupledcon-
itiesofbothGLICandthenAChR.Inparticular,thenAChRin formation.Aromatic-aromaticinteractionscouldplayaroledic-
complexnaturallipidmembranes,suchasthosecomposedof tatingthepropensityofapLGICtoadoptanuncoupledconfor-
eithersoybeanasolectinlipids,ormixturesofdefinedlipids(PC mationinPCmembranes.
pluscholesterolandanioniclipids)adoptsanactivatablerest- In light of this hypothesis, we compared the aromatic-aro-
ing conformation. In contrast, the nAChR in PC membranes maticinteractionsthatexistbetweenM4andM1(cid:1)M3inGLIC
lackingthetwo“activatinglipids”adoptsanuncoupledconfor- versusthenAChR(Fig.6).Consistentwiththeabovehypothe-
mationthatbindsagonistwitharestingstate-likeaffinitybut sis,GLICexhibitsaplethoraofaromatic-aromaticinteractions
does not typically undergo agonist-induced conformational between M4 and M1(cid:1)M3, with clusters located in both the
transitions.WeareinterestedintheuncouplednAChRbecause cytoplasmicandextracellularleafletsofthelipidbilayer.These
of increasing anecdotal evidence hinting that functionally aromaticscouldleadtoahighlystabletransmembranedomain
uncouplednAChRsmayplayaroleinnicotinicreceptorphys- thatisresistanttotheformationofanuncoupledconformation.
APRIL19,2013•VOLUME288•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 11301
LipidSensitivityofaProkaryoticpLGIC
FIGURE6.Aromatic-aromaticinteractionsmaydictatethepropensityofapLGICtoadoptalipid-dependentuncoupledconformation.Shownisa
comparisonofthearomaticresidueslocatedinM1,M3,andM4ofasinglesubunittransmembranedomainforGLIC(A)andthe(cid:1)-subunitofthenAChR(B).Both
asideviewofthetransmembranedomainofeachsubunitandatopviewlookingdownatthebilayersurfaceareshown.Notethatthesidevieworientations
ofthetwotransmembranedomainshavebeenrotated180oaboutthelongaxisofeachmoleculerelativetothesidevieworientationshownintheschematic
diagramofuncouplinginsupplementalFig.S6,becausethispresentsaclearerviewofthearomatic-aromaticinteractionsattheinterfacebetweenM4and
M1(cid:1)M3.
Incontrast,whereasthenAChRexhibitsaromaticresiduesat into the molecular coupling of pLGICs with their membrane
theinterfacebetweenM4andM1(cid:1)M3,thereappeartobefew environment.
if any direct interactions that could stabilize the nAChR in a
coupled conformation where M4 binds tightly to M1(cid:1)M3, Acknowledgments—WethankJulianSurujbaliandPeterJurankafor
although the lower resolution of the nAChR structure makes assistancewiththeelectrophysiologicalmeasurements.
thisinterpretationlessdefinitive.Althoughotherfeatureslikely
playimportantroles,thepaucityofaromatic-aromaticinterac-
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