Gap Junction Channels Gap Junction Channels Presented by: Ima Ima - PowerPoint PPT Presentation

Gap Junction Channels Gap Junction Channels Presented by: Ima Ima Student Student Presented by:

Overview Overview � Intracellular communication Intracellular communication � � Human touch Human touch � – Autosomal recessive deafness Autosomal recessive deafness – – X X- -linked Charcot linked Charcot- -Marie Marie- -Tooth disease Tooth disease – – Cx32 congential demylinated neuropathy Cx32 congential demylinated neuropathy – – Cx50 congential cataracts Cx50 congential cataracts – � Transgenic mice lacking Transgenic mice lacking α α 1 Cx43 1 Cx43 � – Principle heart gap junction Principle heart gap junction –

Three- -Dimensional Structure Dimensional Structure Three of a Recombinant Gap of a Recombinant Gap Junction Membrane Channel Junction Membrane Channel Vinzenz M. Unger, Nalin M. Kumar, Vinzenz M. Unger, Nalin M. Kumar, Norton B. Gilula, Mark Yeager Norton B. Gilula, Mark Yeager

Objective Objective � 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 α α helices. helices. 2 rings of � Wild type and mutant connexins Wild type and mutant connexins � expressed in BHK cells expressed in BHK cells – α α 1 -Cx263T mutant Cx263T mutant – 1 -

Table 1 Table 1 Resolution Resolution � ~ 7.5 ~ 7.5 Å Å in in � membrane plane membrane plane � 21 21 Å Å vertical vertical � Range of tilt Range of tilt � 0 0° ° to 35.3 to 35.3° ° �

Figure 1: Phase and Figure 1: Phase and Amplitude Amplitude

Figure 2a Figure 2a � Tripartite Tripartite � arrangement arrangement � ~150 ~150 Å Å thick thick � � “ “M M” ” outer outer � diameter ~70 Å Å diameter ~70 � “ “E E” ” outer outer � diameter ~50 Å Å diameter ~50

Figure 2b Figure 2b � Vertical section Vertical section � � Narrowing 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 Å Å � Center diameter Center diameter � ~25 Å Å ~25

Figure 2 Figure 2

Figure 2c Figure 2c � Red contours: 1 Red contours: 1 σ σ � above mean density; above mean density; resolution 15 Å Å resolution 15 � Yellow contours: Yellow contours: � 1.5 σ σ above mean; above mean; 1.5 resolution 17.5 Å Å resolution 17.5 � 24 TM helices per 24 TM helices per � connexin, 48 per connexin, 48 per channel. channel.

Figure 3a: Helical packing Figure 3a: Helical packing arrangement. arrangement.

Figure 3b Figure 3b � C tilt and C tilt and � narrowing of the narrowing of the pore. pore. � C & B line the pore C & B line the pore � � Cytoplasmic Cytoplasmic � connections connections between helices? between helices?

Figure 3 Figure 3

Figure 4 Figure 4 Possible subunit boundaries boundaries Possible subunit

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. Nicholson Nicholson

Objective Objective � Identify pore Identify pore- -lining residues lining residues � – SCAM SCAM – � Determine the pore lining helices Determine the pore lining helices � � Make helical assignments Make helical assignments � – Topology map Topology map ⇒ ⇒ 3D model 3D model –

SCAM SCAM � Channel lumen facing protein domains Channel lumen facing protein domains � � Substitute cysteine for one a.a. at a Substitute cysteine for one a.a. at a � time in domains. time in domains. � Add aqueous thiol reagent Add aqueous thiol reagent � � Measure conductance through channel Measure conductance through channel � � Perform for open and closed states of Perform for open and closed states of � the channel. the channel.

Figure 1: Paired oocyte Figure 1: Paired oocyte perfusion system. perfusion system.

Mutants of Cx32 Mutants of Cx32 � 48 mutants total 48 mutants total � � 3 Nonfunctional: W77C, W133C, 3 Nonfunctional: W77C, W133C, � T134C T134C � 36 Minimal changes in conductance 36 Minimal changes in conductance � (candidates) (candidates) � 7 7 “ “Reverse Reverse- -gating gating” ” mutants mutants � � Cx32E146C Cx32E146C � � Cx32A88C Cx32A88C �

Figure 2 Figure 2 � Candidates Candidates � ⊗ Nonfunctional Nonfunctional ⊗ � “ “Reverse Reverse � gating” ” gating � Altered Altered � channel channel properties properties

Figure 3: A- -C C Figure 3: A Wild type Representative candidate mutants

Figure 3: A & D Figure 3: A & D Wild type “Reverse-gating” (heterotypic)

Figure 3F: Cx32E146C Figure 3F: Cx32E146C � Nonfunctional Nonfunctional � channel channel homotypically or homotypically or heterotypically with heterotypically with wtCx32 wtCx32 � Add DTT to mutant: Add DTT to mutant: � wt junctional wt junctional current restored. current restored.

Disulfide bond between Disulfide bond between E146C & C201 E146C & C201

Cx32A88C Cx32A88C � Lethal to oocytes Lethal to oocytes � � 10 fold increase in membrane 10 fold increase in membrane � conductance conductance � Current characteristic of open Current characteristic of open � hemichannels hemichannels

Figure 4: Perfused Figure 4: Perfused oocytes oocytes 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.

Figure 5A: MBB Figure 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. �

Figure 5B: Western Blot Figure 5B: Western Blot � Lane 1: Cx32 from Lane 1: Cx32 from � intact oocytes intact oocytes � Lane 2: Cx32 Lane 2: Cx32 � perfused with MBB perfused with MBB � Lane 3: Noninjected Lane 3: Noninjected � oocytes exposed to oocytes exposed to MBB MBB � Lane 4: Perfused Lane 4: Perfused � Cx32 not exposed Cx32 not exposed to MBB to MBB

Figure 5: C & D Figure 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.

Figure 6 Figure 6

� Majority of reactive Majority of reactive � residues on M3 residues on M3 � M1: 4 M1: 4 “ “reverse reverse- - � gating” ” reactive reactive gating residues residues � M2: reactive near M2: reactive near � cytoplasmic end or cytoplasmic end or proline proline � M4: reactive near M4: reactive near � extracellular end extracellular end

Non- -pore lining cystines pore lining cystines Non � From structure From structure � model only 2 model only 2 helices thought to helices thought to line the pore. line the pore. � Aqueous Aqueous “ “crevices crevices” ”? ? �

Figure 7 Figure 7 Reactive sites eliminated as pore lining sites.

Figure 8: Summary Figure 8: Summary

Summary Summary � M2 & M3 (A & C) are the pore M2 & 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 residues Helical periodicity of reactive residues � � Towards extracellular end reactivity of Towards extracellular end reactivity of � β - -sheet pattern sheet pattern β � F149C highest block reactivity F149C highest block reactivity � – Narrowing point of M3 Narrowing point of M3 –

Figure 9 Figure 9 M1 = B M2 = A M3 = C M4 = D M1 = B M2 = A M3 = C M4 = D Open state Partial closed state

Figure 10 Figure 10

Discussion Discussion � 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? �