Gap Junction Channels - University of Colorado...
Transcript of Gap Junction Channels - University of Colorado...
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Gap Junction ChannelsGap Junction Channels
Presented by: Presented by: ImaIma StudentStudent
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OverviewOverview
Intracellular communicationIntracellular communicationHuman touchHuman touch–– Autosomal recessive deafnessAutosomal recessive deafness–– XX--linked Charcotlinked Charcot--MarieMarie--Tooth diseaseTooth disease–– Cx32 congential demylinated neuropathyCx32 congential demylinated neuropathy–– Cx50 congential cataractsCx50 congential cataractsTransgenic mice lacking Transgenic mice lacking αα11 Cx43Cx43–– Principle heart gap junction Principle heart gap junction
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ThreeThree--Dimensional Structure Dimensional Structure of a Recombinant Gap of a Recombinant Gap
Junction Membrane ChannelJunction Membrane Channel
Vinzenz M. Unger, Nalin M. Kumar, Vinzenz M. Unger, Nalin M. Kumar, Norton B. Gilula, Mark YeagerNorton B. Gilula, Mark Yeager
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ObjectiveObjective
3D analysis to explore transmembrane 3D analysis to explore transmembrane architecture.architecture.Previously 2D crystal analysis suggest Previously 2D crystal analysis suggest 2 rings of 2 rings of αα helices.helices.
Wild type and mutant connexins Wild type and mutant connexins expressed in BHK cellsexpressed in BHK cells–– αα11--Cx263T mutantCx263T mutant
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Table 1Table 1
ResolutionResolution~ 7.5 ~ 7.5 ÅÅ in in membrane planemembrane plane21 21 ÅÅ verticalvertical
Range of tiltRange of tilt00°° to 35.3to 35.3°°
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Figure 1: Phase and Figure 1: Phase and AmplitudeAmplitude
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Figure 2aFigure 2a
Tripartite Tripartite arrangementarrangement~150 ~150 ÅÅ thickthick““MM”” outer outer diameter ~70 diameter ~70 ÅÅ““EE”” outer outer diameter ~50 diameter ~50 ÅÅ
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Figure 2bFigure 2b
Vertical sectionVertical sectionNarrowing of Narrowing of channel occurs channel occurs when crossing the when crossing the lipid bilayer.lipid bilayer.–– From 40 to 15 From 40 to 15 ÅÅ
Center diameter Center diameter ~25 ~25 ÅÅ
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Figure 2Figure 2
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Figure 2cFigure 2c
Red contours: 1Red contours: 1σσabove mean density; above mean density; resolution 15 resolution 15 ÅÅYellow contours: Yellow contours: 1.51.5σσ above mean; above mean; resolution 17.5 resolution 17.5 ÅÅ24 TM helices per 24 TM helices per connexin, 48 per connexin, 48 per channel.channel.
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Figure 3a: Helical packing Figure 3a: Helical packing arrangement.arrangement.
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Figure 3bFigure 3b
C tilt and C tilt and narrowing of the narrowing of the pore.pore.C & B line the poreC & B line the poreCytoplasmic Cytoplasmic connections connections between helices?between helices?
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Figure 3Figure 3
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Figure 4Figure 4
Possible subunitPossible subunit boundariesboundaries
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Identification of amino acid Identification of amino acid residues lining the pore of a residues lining the pore of a
gap channel.gap channel.
I.M. Skerrett, J. Aronowitz, J.H. Shin, I.M. Skerrett, J. Aronowitz, J.H. Shin, G. Cymes, E. Kasperek, F.L. Cao, B.J. G. Cymes, E. Kasperek, F.L. Cao, B.J. NicholsonNicholson
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ObjectiveObjective
Identify poreIdentify pore--lining residueslining residues–– SCAMSCAM
Determine the pore lining helicesDetermine the pore lining helicesMake helical assignmentsMake helical assignments–– Topology map Topology map ⇒⇒ 3D model3D model
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SCAMSCAM
Channel lumen facing protein domainsChannel lumen facing protein domainsSubstitute cysteine for one a.a. at a Substitute cysteine for one a.a. at a time in domains.time in domains.Add aqueous thiol reagentAdd aqueous thiol reagentMeasure conductance through channelMeasure conductance through channelPerform for open and closed states of Perform for open and closed states of the channel. the channel.
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Figure 1: Paired oocyte Figure 1: Paired oocyte perfusion system.perfusion system.
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Mutants of Cx32Mutants of Cx32
48 mutants total48 mutants total3 Nonfunctional: W77C, W133C, 3 Nonfunctional: W77C, W133C, T134CT134C36 Minimal changes in conductance 36 Minimal changes in conductance
(candidates)(candidates)7 7 ““ReverseReverse--gatinggating”” mutantsmutantsCx32E146CCx32E146CCx32A88CCx32A88C
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Figure 2Figure 2
CandidatesCandidates⊗⊗ NonfunctionalNonfunctional
““Reverse Reverse gatinggating””Altered Altered channel channel propertiesproperties
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Figure 3: AFigure 3: A--CC
Wild type Representative candidate mutants
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Figure 3: A & DFigure 3: A & D
Wild type “Reverse-gating”
(heterotypic)
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Figure 3F: Cx32E146CFigure 3F: Cx32E146C
Nonfunctional Nonfunctional channel channel homotypically or homotypically or heterotypically with heterotypically with wtCx32wtCx32Add DTT to mutant: Add DTT to mutant: wt junctional wt junctional current restored.current restored.
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Disulfide bond between Disulfide bond between E146C & C201E146C & C201
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Cx32A88CCx32A88C
Lethal to oocytesLethal to oocytes10 fold increase in membrane 10 fold increase in membrane conductanceconductanceCurrent characteristic of open Current characteristic of open hemichannelshemichannels
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Figure 4: Perfused Figure 4: Perfused oocytesoocytes
Minimal changes in transjunctional current when Minimal changes in transjunctional current when oocytes are cut and perfused. Partial loss of Vj oocytes are cut and perfused. Partial loss of Vj sensitivity.sensitivity.
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Figure 5A: MBBFigure 5A: MBB
Large thiol reagent, still too small to cause Large thiol reagent, still too small to cause full channel blockage.full channel blockage.Maleimide is an irreversible thiol reagent.Maleimide is an irreversible thiol reagent.
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Figure 5B: Western BlotFigure 5B: Western Blot
Lane 1: Cx32 from Lane 1: Cx32 from intact oocytesintact oocytesLane 2: Cx32 Lane 2: Cx32 perfused with MBBperfused with MBBLane 3: Noninjected Lane 3: Noninjected oocytes exposed to oocytes exposed to MBBMBBLane 4: Perfused Lane 4: Perfused Cx32 not exposed Cx32 not exposed to MBBto MBB
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Figure 5: C & DFigure 5: C & D
Cx32 wt: conduction Cx32 wt: conduction increases before & increases before & after treatment.after treatment.Reactive mutants: Reactive mutants: conduction decreases conduction decreases after treatment.after treatment.
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Figure 6Figure 6
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Majority of reactive Majority of reactive residues on M3residues on M3M1: 4 M1: 4 ““reversereverse--gatinggating”” reactive reactive residuesresiduesM2: reactive near M2: reactive near cytoplasmic end or cytoplasmic end or prolineprolineM4: reactive near M4: reactive near extracellular endextracellular end
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NonNon--pore lining cystinespore lining cystines
From structure From structure model only 2 model only 2 helices thought to helices thought to line the pore.line the pore.
Aqueous Aqueous ““crevicescrevices””??
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Figure 7Figure 7
Reactive sites eliminated as pore lining sites.
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Figure 8: SummaryFigure 8: Summary
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SummarySummary
M2 & M3 (A & C) are the poreM2 & M3 (A & C) are the pore--lining lining domains in the open state. domains in the open state. –– Not M1 (B)Not M1 (B)Helical periodicity of reactive residuesHelical periodicity of reactive residuesTowards extracellular end reactivity of Towards extracellular end reactivity of ββ--sheet patternsheet patternF149C highest block reactivityF149C highest block reactivity–– Narrowing point of M3Narrowing point of M3
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Figure 9Figure 9M1 = B M2 = A M3 = C M4 = DM1 = B M2 = A M3 = C M4 = D
Open state Partial closed state
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Figure 10Figure 10
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DiscussionDiscussion
Why have a pore that is partially open Why have a pore that is partially open all the time?all the time?Preference of cations over anions.Preference of cations over anions.Why preference of ATP over ADP?Why preference of ATP over ADP?