What the cryoEM structure of the InsP 3 receptor

2. 25Å What the cryoEM structure of the InsP3 receptor can tell us about intracellular calcium signaling Barbara Ehrlich Pharmacology June 10, 2005

3. Calcium

4. Calcium is stored in the endoplasmic reticulum CGB

5. 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

6. InsP3R are concentrated in cerebellar Purkinje cells Rat cerebellar slice [12 days PN] . Polyclonal antibody for type I InsP3 receptor, Llano and Ehrlich

7. 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

8. 250 100 50 Popen 0% 2% The InsP3R is a large protein that makes an ion channel kDa

9. Popen 0% 2% Chromogranin B (CGB) Addition of a protein partner and an analog of InsP3 allows the InsP3R to remain open 0.5 mM ATP 0.5 mM Ca2+ 100 nM AdA 1.0 mg/ml CGB 96% Adenophostin (AdA)

10. q, y q, y 0.0, 0.0 4.8, 0.0 48.0, 7.5 33.6, 20.0 57.6, 38.6 57.6, 45.0 76.8, 45.0 67.2, 54.0 86.4, 33.8 86.4, 39.4 Examples of particles used for 3D reconstruction Match between model projections and class averages

11. CryoEM structure of the InsP3R using two analysis protocols InsP3R Closed State 24 A resolution Jiang et al. (2002) Current Closed State

12. The InsP3R in the closed and open state closed open

13. Low high 25Å Cytosol Membrane Lumen 50 Å The InsP3 binding domain fits on the “arms” CLOSED

14. Low high CLOSED OPEN AdA, ATP, Ca2+ Cytosol Membrane CGB4 Lumen 50 Å 50 Å CGB4 The InsP3R shape changes when it opens

15. 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

16. InsP3R control schizophrenia normal dendrites cell body bile duct obstruction hepatitis C 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 Nowakowski et al, Schizophrenia Research 58 (2002) 43-53 Hirata et al, Gastroenterology 121 (2002) 1088-1100

17. CGB4 50 Å 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?

18. Mike Nathanson Juliana Rengifo Bill Grenawitzke David Chester Chi-un Choe Edwin Thrower Qiu-Xing Jiang Fred Sigworth Nils Nicolay Brenda DeGray Daniel Hertle Mark Yeckel Collaborators

20. Comparison of the intracellular calcium release channels InsP3R type l RyR type 1

21. NCS-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 Johenning et al, 2002

22. Intracellular calcium signaling model

23. The amplitude of the calcium transient can be modulated Dolmetsch, Xu and Lewis, 1998

24. The frequency of the calcium oscillation can be modulated Dolmetsch, Xu and Lewis, 1998

25. InsP3 R 1 CGB InsP3R type I InsP3R type III Not all InsP3R are alike Different functional properties Different location in cells Found in different cells CA3 CA1 CA3 CA1 CA3 CA1