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

Gap Junction Channels Gap Junction Channels

Presented by: Presented by: Ima Ima Student Student

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 α1

1 Cx43

Cx43

– – Principle heart gap junction Principle heart gap junction

Three Three-

• Dimensional Structure

Dimensional Structure

• f a Recombinant Gap
• f 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 2 rings of α α helices. helices.

• Wild type and mutant connexins

Wild type and mutant connexins expressed in BHK cells expressed in BHK cells

– – α α1

1-

• Cx263T mutant

Cx263T mutant

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°

° 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

• uter

diameter ~70 diameter ~70 Å Å

• “

“E E” ” outer

• uter

diameter ~50 diameter ~50 Å Å

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 ~25 Å Å

Figure 2 Figure 2

Figure 2c Figure 2c

• Red contours: 1

Red contours: 1σ σ above mean density; above mean density; resolution 15 resolution 15 Å Å

• Yellow contours:

Yellow contours: 1.5 1.5σ σ above mean; above mean; resolution 17.5 resolution 17.5 Å Å

• 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 Possible subunit boundaries boundaries

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 Figure 3: A-

• C

C

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

• ocytes
• ocytes

Minimal changes in transjunctional current when Minimal changes in transjunctional current when

• ocytes are cut and perfused. Partial loss of Vj
• ocytes 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

• ocytes exposed to
• ocytes 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

gating” ” reactive reactive 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 Non-

• pore lining cystines

pore 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 “ “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 β-

• sheet pattern

sheet pattern

• 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?