1. Gene expression, LTP, ion channels, enzymes, cell death
Calcium as a central player in cell signaling
Gene expression, LTP, ion channels, enzymes, cell death

2. How do we measure Ca2+ in a cell?
Biological response:
muscle shortening
secretion
enzyme activity
K+ channel current
Radioactive 45Ca2+
Fluorescent dyes
Genetically encoded calcium indicators

3. Fura-2 and GCaMP2 signals evoked by 5HT in ASMCs transfected with α2(f)GCaMP2. (A) DsRed2, GCaMP2, and fura-2 images. (Scale bar: 20 μm.) (B) Time course of Ca2+ signals detected by fura-2 (Upper) and GCaMP2 (Lower) within the blue and green boxes in the fura-2 image in A. (C) Pseudocolor fura-2 ratio (Upper) and GCaMP2 fluorescence (Lower) images from the same field as in A. a and b were captured at time points “a” and “b” indicated on the graphs in B. Relative color scale indicates low (L) and high (H) [Ca2+]. Similar results were obtained in >20 such experiments.
Lee M Y et al. PNAS 2006;103:
©2006 by National Academy of Sciences

4. gCAMP in drosophila brain
gCAMp in mushroom bodies (fly brain processing center) bottom. Top is primary cell culture before and after stimulation. gCAMp is a fusion between GFP and the Ca binding site of calmodulin.

5. Life cycle of calcium tightly regulated
Life cycle of calcium tightly regulated. Focus on where calcium is stored, where it comes in where it goes out. Proteins responsible for keeping concentrations in the same range despite local perturbances.

6. Non-uniform distribution of calcium
Non-uniform distribution of calcium. Total Ca=11mM, free calcium is about 100nM (1:10,000 ratio)

7. Entry of Calcium L-type Ca channel Long-lasting High voltage activated
In HPC cells, found on
apical dendrites
N-type Ca channel
High voltage activated
Found at pre-synapse
Necessary for exocytosis

8. L-type VACC mediate glutamate induced Ca2+ influx
Nimodipine is an L-type calcium channel blocker. Used to treat cardiac arrythmia

9. Efflux of Calcium Calcium Pump Na/Ca exchanger
Transport of calcium at membranes. Keep ca low inside. Buffering mechanisms….
Calcium pump 1 Ca for 1 ATP, high affinity, low capacity (sufficient for cells at rest)
Exchanger 3Na for 1 Ca, bidirectional. At more negative Vm, Na enters, at less negative Vm, Ca will enter. Lower affinity, larger (50X) capacity.
Calcium binding proteins
VACC

10. ER- SERCA pumps Ca in. Specialized ER, contains calcium binding proteins, calsequestrin in muscle, calreticulin in neurons.
Calcium in ER facilitates chaperone activities—protein folding
Cytoplasmic buffering to keep concentration low
Allow release (IP3 stimulated) upon signal

11. Alzheimer’s disease proteins, presenilins, regulate SERCA activity
PS1 and PS2 mutations cause AD, modeled in mice by knocking these genes out, singly or together (DKO)
Thapsigargin was applied to inhibit SERCA—so how much Ca is released reflects how much was stored in the ER (less in PS KO)

12. Elevated cytosolic Ca2+ and reduced ER Ca2+ stores in presenilin-null cell line.
Elevated cytosolic Ca2+ and reduced ER Ca2+ stores in presenilin-null cell line. (A) Changes in cytosolic Ca2+ evoked by thapsigargin in control fibroblasts (control; n = 48 cells) and fibroblasts from PSDKO (n = 57 cells), PS1KO (n = 48), and PS2KO (n = 56) mice. ER Ca2+ stores were released into the cytosol by application of 1 μM thapsigargin, a specific blocker of SERCA activity, with 0 mM Ca2+ in the bathing solution. Basal cytosolic Ca2+ levels were elevated (∼70 nM) in the PSDKO and PS2KO fibroblasts compared with controls and PS1KO cells (∼50 nM; P < 0.05), whereas the peak Ca2+ signals after application of thapsigargin were substantially reduced in PSDKO and PS2KO fibroblasts. (B) Mean values of basal cytosolic [Ca2+] and thapsigargin-evoked Ca2+ signals, derived from the experiments in A. *, significance in peak rise versus pcDNA (P < 0.05); #, significance in basal levels versus pcDNA (P < 0.05). (C) Changes in cytosolic Ca2+ evoked by 1 μM ionomycin in control fibroblasts (control; n = 12 cells) and fibroblasts from PSDKO mice (n = 37 cells), with 0 mM Ca2+ in the bathing solution. (D) Cytosolic Ca2+ signals in PSDKO fibroblasts transfected either with pcDNA (n = 63 cells) or AICD (n = 54 cells) 48 h earlier. No significant differences were apparent in either basal cytosolic Ca2+ levels or the peak response after application of 1 μM thapsigargin. Error bars show SEM.
Kim N. Green et al. J Cell Biol 2008;181:
© 2008 Green et al.

13. Accessing Ca2+ from the ER
Calcium at the endoplasmic reticulum. In via SERCA-like pumps, out via IP3 or ryanodine receptors. Both are homotetramers and quite similar to each other. Ryanodine receptor is also a calcium induced calcium release channel (CICR). IP3 receptors are activated by low calcium, but inhibited by high calcium in the cytoplasm.

14. CICR in excitable cells
Excitable cells: VACC Ca induces release from ryanodine receptor (CICR): much larger than IP3 induced release

15. Mitochondria buffer calcium at higher concentrations, but capacity is higher than ER

16. Local calcium around mitochondria may reach threshold
Local calcium around mitochondria may reach threshold. Especially when mitochondria are right next to ER stores. Differential sensitivity to uptake. cADPR is an activator of ryanodine receptor (CICR).

17. Family of calcium binding proteins with diverse cellular functions
Family of calcium binding proteins with diverse cellular functions. dysregulation of any of these genes contributes to many neurodegenerative diseases.

18. Calcium buffering proteins
Calbindin, red neurofilament
A CB +PV
B CB +CR
CB red in both

19. Effect of buffering on calcium transients
Different

20. Calcium waves in astrocytes

21. Calcium in biological processes.