What the cryoEM structure of the InsP3 receptor
can tell us about intracellular calcium signaling
25Å
Barbara Ehrlich
Pharmacology
June 10, 2005
Calcium
Calcium is stored in the endoplasmic reticulum
CGB
Changes in intracellular calcium signaling
occur in many diseases
Huntington’s Disease
Alzheimer’s Disease
Parkinson’s Disease
Schizophrenia
Heart Failure
Bile Duct Obstruction
Polycystic Kidney Disease
InsP3R are concentrated
in cerebellar Purkinje cells
Rat cerebellar slice [12 days PN] . Polyclonal antibody for type I InsP3 receptor, Llano and Ehrlich
InsP3R domains
Ligand-Binding
Domain
N
Regulatoy
Domain
C
Channel
Domain
Patel S, Joseph SK, Thomas AP.
Cell Calcium 1999 Mar;25(3):247-64
The InsP3R is a large protein
that makes an ion channel
kDa
250
100
50
Popen
0%
2%
Addition of a protein partner and an analog of InsP3
allows the InsP3R to remain open
Popen
0%
2%
Chromogranin B (CGB)
Adenophostin (AdA)
0.5 mM ATP
0.5 mM Ca2+
100 nM AdA
1.0 mg/ml CGB
96%
Examples of particles used
for 3D reconstruction
Match between model projections
and class averages
q, y
q, y
0.0, 0.0
4.8,
0.0
33.6, 20.0
48.0, 7.5
57.6, 38.6
57.6, 45.0
67.2, 54.0
76.8, 45.0
86.4, 33.8
86.4, 39.4
CryoEM structure of the InsP3R
using two analysis protocols
InsP3R Closed State
24 A resolution
Jiang et al. (2002)
Current Closed State
The InsP3R in the closed and open state
closed
open
The InsP3 binding domain fits on the “arms”
25Å
CLOSED
Low
high
Cytosol
Membrane
50 Å
Lumen
The InsP3R shape changes when it opens
CLOSED
OPEN
Low
AdA, ATP, Ca2+
high
Cytosol
Membrane
50 Å
CGB4
Lumen
50 Å
CGB4
What we have learned already
Structure changes during activity
surprisingly large conformational changes
suggests there is a mechanical gate
Good idea where InsP3 binds
the shape of the binding pocket will suggest
classes of compounds to test as
agonists and antagonists
Disease-related changes in calcium signaling
Chromogranin B levels are
lower in brain tissue from
patients with schizophrenia
Loss of InsP3R from bile duct
epithelia is found in diseases
that block the bile duct
InsP3R
control
schizophrenia
dendrites
normal
cell body
bile duct
obstruction
hepatitis C
Nowakowski et al,
Schizophrenia Research 58 (2002) 43-53
Hirata et al,
Gastroenterology 121 (2002) 1088-1100
What we hope to learn
Can we design new channel openers and closers?
Where do the regulatory factors bind?
Can these regulatory proteins be used
as pharmacological agents?
Can the structure tell us why the InsP3Rs cluster
and go to distinct places in the cell?
50 Å
CGB4
Collaborators
Juliana Rengifo Bill Grenawitzke
Chi-un Choe
Nils Nicolay
Qiu-Xing Jiang Fred Sigworth
Edwin Thrower
David Chester
Brenda DeGray
Mike Nathanson
Daniel Hertle
Mark Yeckel
Comparison of the intracellular calcium release channels
InsP3R type l
RyR type 1
The localization of CGB is not uniform in PC12 cells
NCS-1 is uniform
CGB could be a factor in determining the signal initiation site
Chromogranin B
NCS-1
Johenning et al, 2002
Intracellular calcium signaling model
The amplitude of the calcium transient can be modulated
Dolmetsch, Xu and Lewis, 1998
The frequency of the calcium oscillation can be modulated
Dolmetsch, Xu and Lewis, 1998
Not all InsP3R are alike
Different functional properties
Different location in cells
Found in different cells
CA3
InsP3 R 1
InsP3R type I
CA1
InsP3R type III
CGB
CA3
CA1
CA3
CA1