1. cAMP and Calcium Signaling Pathways are Often Interdependent 1. cAMP and PKA regulated calcium channels 2. Calcium regulated adenylyl cyclases and phosphodiesterases Calcium Signaling and Measurement

2. Chapter 3 Figure 14

3. Calcium signals can be very local! 400 200 0 500 500 nm 0 Ca i (  M) Spatial profiles of intracellular free Ca 2+ calculated assuming that 10 Ca 2+ channels are open. Entering Ca 2+ ions diffuse but soon bind to mobile and immobile Ca 2+ buffers. The concentration profiles are very steep, and only around the channels are there many unbound ions. (Lenzi & Roberts, 1994) distance Amazing! distance concentration Ca i

4. Source: Neuron, Volume 73, Issue 5, Pages 862-885 (DOI:10.1016/j.neuron.2012.02.011)Neuron, Volume 73, Issue 5, Pages 862-885 Copyright © 2012 Elsevier Inc. Terms and ConditionsTerms and Conditions

8. .

11. Andreasen et al (1983): Purification of a Novel Calmodulin Binding Protein, Neuromodulin, from Bovine Cerebral Cortex Membranes. Biochem. 22:4615-4618. 1. Neuromodulin is an abundant calmodulin binding protein that is localized in growth cones and synapses of growing neurons. 2. Neuromodulin binds calmodulin in the absence of calcium and localizes calmodulin within growth cones and synapses. 3. Neuromodulin releases calmodulin when calcium goes up and when it is phosphorylated within its IQ domain by PKC, the site for calmodulin binding It is hypothesized that neuromodulin binds and localizes calmodulin at specific sites in the cell and release free calmodulin locally when calcium increases and PKC is activated. Neuromodulin (GAP-43, P-57) Binds Calmodulin in the Absence of Calcium

12. Xia and Storm (2005) Nature Rev Neurosci 6:267-276

14. Laporte et al., (1980) Biochem 19:3814-9

15. Calmodulin Structure

17. Table I. Some CaM-Regulated Proteins Enzymes Cyclic nucleotide phosphodiesterases (e.g. the PDE1 family) Adenylyl cyclases (AC1 and AC8) ATP-dependent Ca 2+ pumps Myosin light chain kinase CaM kinase I, II and IV Phosphorylase kinase Calcineurin (a protein phosphatase) NO synthase RasGRF1, a CaM-activated GEF Nonenzymatic proteins Tubulin Troponin I Spectrin, fodrin, caldesmon, calspectin, cytosynalin MAP-2 and Tau Neuromodulin and Neurogranin Ca 2+ channels

18. The Affinity of Calmodulin for Calcium is Enhanced by Troponin I Keller et al., (1982) Biochem. 21:156

19. The Free Energy of Coupling for Calmodulin Binding to Target Proteins Varies with Each Protein Olwin et al (1984) J. Biol. Chem. 259:10949

20. The Calcium binding Curves to Calmodulin Depend on Which Calmodulin Binding Protein is Present

22. Figure 2 Source: Neuron, Volume 73, Issue 5, Pages 862-885 (DOI:10.1016/j.neuron.2012.02.011)Neuron, Volume 73, Issue 5, Pages 862-885 Copyright © 2012 Elsevier Inc. Terms and ConditionsTerms and Conditions

23. 340 or 380 nm (UV) 510 nm Fura-2 excitation emission UV excitation wavelength (nm) Grynkeiwicz, Poenie, Tsien (1985) 300 400 1 mM 1.3 uM 600 nM 350 nM 230 nM 150 nM ~ K D 100 nM 66 nM 38 nM 17 nM 0 nM 340 nm380 nm Test solutions Ca 2+ -bound Ca 2+ -free Fluorescence intensity 0 2 4 Fura-2: a ratiometric Ca 2+ indicator dye for cells Slide from conj531, Hille Why ratio? Measure @ 510 Ideally 1st subtract "nonspecific fluoresence from each

24. Figure 3 Source: Neuron, Volume 73, Issue 5, Pages 862-885 (DOI:10.1016/j.neuron.2012.02.011)Neuron, Volume 73, Issue 5, Pages 862-885 Copyright © 2012 Elsevier Inc. Terms and ConditionsTerms and Conditions

25. Figure 4 Source: Neuron, Volume 73, Issue 5, Pages 862-885 (DOI:10.1016/j.neuron.2012.02.011)Neuron, Volume 73, Issue 5, Pages 862-885 Copyright © 2012 Elsevier Inc. Terms and ConditionsTerms and Conditions

26. Glucagon stimulation of Ca2+ oscillations in HEK-293 cells expressing the glucagon receptor and III-AC (Fura-2 AM loading) Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

27. Representative Ca2+ responses to glucagon in III-AC-G cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

28. Isoproterenol stimulation of Ca2+ oscillations in HEK-293 cells expressing III-AC. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

29. Forskolin stimulation of Ca2+ oscillations in HEK-293 cells expressing III-AC. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

30. Inhibitors of cAMP-dependent protein kinase block hormone-stimulated Ca2+ oscillations in III-AC-G cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

31. Effect of dibutyryl cAMP and (Sp)-cAMP on intracellular free Ca2+ in HEK-293 cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

32. Ryanodine does not affect glucagon-stimulated Ca2+ oscillations in III-AC-G cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

33. Extracellular Ca2+ is not required for hormone-stimulated Ca2+ oscillations in III-AC-G cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

34. Hormone-stimulated Ca2+ oscillations in III-AC-G cells are blocked by the CaM kinase inhibitor KN-62. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

35. Mechanism for hormone-stimulated Ca2+ oscillations in III-AC-G cells. Wayman G A et al. J. Biol. Chem. 1995;270:24108-24115 ©1995 by American Society for Biochemistry and Molecular Biology

42. Sindreau et al., unpublished observations Detection of Calcium Transients Caused by Kanic Acid Injection into, the Hippocampus

43. Time-Lapse of Hippocampal Neural Activation During Learning & Memory Sindreu et al; unpublished data Context Retrieval Home cage Pre-shock Shock Retrieval GCaMP3 Fluorescence (single cell in dentate gyrus) Time (s) shock retrieval

44. GWAS have Identified AC3 as one of top-ranked target gene.

45. Basal CA3-CA1 synaptic activities are reduced in hippocampal slices from AC3 KO mice.

46. AC3 -/- mice have much smaller pop-spikes in CA1 layer than that of AC3 +/+ mice

47. CA1 neurons of AC3 -/- mice demonstrated reduced excitation in response to foot shock stimulation. Chen et al., unpublished observations