Calcium Signaling and its Implication in Disease

Calcium Signalling:

Import and export help in Ca signaling through changing cytosolic Ca concentration, reverted back by pumps.

Import through voltage dependent Ca channels (VDCC), SOCC, NSCC, NCX (exchange), NCKX (co-transport) from extracellular side and exported out of ER and SR via RyR regulated by IP3R. (100nM-1mM).

Voltage gated ca+2 channels export Ca out of cell, SERCA pumps restore Ca into ER, SR.

1. Calcium Signaling Submitted by: Tashvinder Singh PhD Research Scholar Department of Human Genetics and Molecular Medicine Central University of Punjab, Bathinda. [email protected]

2. Introduction Import and export help in Ca signaling through changing cytosolic Ca concentration, reverted back by pumps. Import through voltage dependent Ca channels (VDCC), SOCC, NSCC, NCX (exchange), NCKX (co-transport) from extracellular side and exported out of ER and SR via RyR regulated by IP3R. (100nM-1mM). Voltage gated ca+2 channels export Ca out of cell, SERCA pumps restore Ca into ER, SR. CaN activation (PP2B) and then NFAT by dephosphorylation. cyclosporin, voclosporin target CaN.

3. Protein Kinase C mediated Signalling

4. Phospholipase C mediated Signalling

5. Ca Pathway Cross Signalling

6. Ca Sensors and Adaptors Calmodulin (CaM) - Ubiquitous adaptor protein and specifically bind to Ca. Contain EF Hand domain (helix-loop-helix motif) bind to Ca. Ca bind to 7 O2 atoms (chelate Ca) of aa in bipyramidal pentagonal shape. Binding with Ca induce dimerisation, changes in conformation, remodelling active sites and exposing hydrophobic surfaces. Hydrophobic surfaces wraps around target proteins, increase CaM/Ca binding specificity.

7. S100 and C2 Domain S100 - Ca sensory protein, also containing EF Hand domain (homo or hetero dimers) helps in assembly and disassembly of protein protein complex, binding to Ca triggers hydrophobic surface exposure. C2 Domain - 120 aa stretch, contain 8 antiparallel beta sandwich and form coordinate bond with Ca (bind in a cavity formed by first and final loop). Protein containing this domain translocated to region where Ca/CaM conc. is profound (DAG and IP3 regulated). PIP2 and CaM Switching - Both -vely charged so compete for +ve charged created by K-Ras, ions and EGFR and this is regulated by Ca/CaM complex, PKC phosphorylation, PIP2 hydrolysis.

8. Ca Pumps PMCA - remove Ca out of the cell and SERCA - restore Ca+2 inside the ER, SR making cytosolic Ca conc. ↓. NCX - exchange 3 Na ion with 1 Ca ion and NCKX - cotransport 1 Ca with 1 K in exchange of 4 Na ions making cytosolic Ca conc. ↑. PMCA/SERCA having high affinity low capacity but NCX/NCKX having high capacity low affinity. That means first can retain Ca for longer duration in the cell but latter can make immediate adjustment in the cell. CaM regulate PMCA affinity and ATPase pump rate.

9. Ca Floodgate CaV conducts roughly 10 million Ca/sec, thus decreasing 20,000 fold gradient. Channel’s antenna (helix-turn-helix) import Ca very selectively. Change in voltage (triggered by messengers) moves paddle very quickly which opens the gate and forming “Ca Cage” upon binding with Ca. Intracellular Ca Signalling Low cytosolic Ca conc. opens the RyR channels, flowing Ca out of the ER. High cytosolic Ca conc. block the gating. This is regulated by Ca/CaM/CaMK2, mAKAP, PKA, PR130/CaN, sorcin, spinophilin and their defects results in cardio-arrhythmias.

10. TBP (Transient receptor Potential) CaV induce cell -cell by triggering protein complexes such as SNARE and synaptotagmins. IP3R release Ca out of the ER faster as compared to Ca channeling across blood cell. SOCE is activated when Ca is depleted in ER (sensed by STIM), thus retrieval of Ca into ER by CRAC. STIM along with Orai1 and TRPC1 activates CRAC. Ca/CaM activated CaN activates NFAT by dephosphorylation which regulate chemokine genes on its translocation to nucleus.

11. Ca acts Locally Ca is distributed by SR to nearby muscles where it binds troponin to induce contraction. Ca/CaM activated PLCb cleaves PIP2 into IP3 and DAG. Ga and Gb/g subunits activates PLCb. RTK dimerizes upon ligand binding and activates PLCg. ER-mitochondrial synapse - uptake of Ca in mitochondria triggers ATP production but conc. exceeding normal triggers apoptosis. Ca bound CaM increase the duration of signals with the help of CaMK11 phosphorylation of substrates.

12. Apoptosis MiCA mediated highly selective Ca entry in mitochondria triggers more ATP production. High or excessive ATP production and subsequent ROS production act as defensive toxin when extruded in cytoplasm. Microbial invasion make release cyt c from mitochondrial outer membrane and further process activation of caspases and Ca dependent annexin, Bcl-2 protein translocation. Ca also regulate sperm flagellar shape but high cytosolic Ca level could halt motility.

13. Smooth Muscle Contraction and Relaxation Activated Ca/CaM complex phosphorylate MLCK which phosphorylate light chain of myosin head and activating myosin ATPase activity. MLC phosphatase helps in smooth muscle relaxation. Ca dependent receptor activates endothelial cells adjacent to smooth muscles which release vasoactive agents that helps in releasing NOS3 (NO release). NO works by activating guanylyl cyclase and PKA to lower Ca level. Cadherin and Integrin both bind to Ca and activate downstream signalling molecules such as talin, paxillin and ⍶-actinins. N-Cadherin/b-Catenin interaction with actin cytoskeleton during contractile movement.

14. Role in Alzheimer’s Disease beta amyloid protein - neurotoxic, neurodegeneration. synaptic dysfunction and neuronal loss. Ca is influx in high amount through VDCC or TRP/Orai1 channel during alzheimer development and beta amyloid seen to interact with these channels. Its hallmarks are - presence of b-amyloid in the form of plaques presence of intracellular filamentous microtubule networks (tau’s aggregates) Nixon et al showed that Ca signalling induce neurofibrillary tangles when signal transduction is hyperactive. increase of Ca activates Ca/CaM/CaMK2 complex that can cross link with tau protein.

15. Role in ALS 5-10 % inherited from parents with mutation in SOD1 gene. SOD1 protein localised to outer mitochondrial membrane and involved in apoptotic signalling and oxidative stress such as mitochondrial death pathway and myocyte apoptotic signalling. Altered Ca signalling pathway accounts for 3 inter-related toxic pathways which are mitochondrial dysfunction, oxidative stress and neuroinflammation. (Manoj Kumar Jaiswal)

16. Djlila Mekahli et. al. - Endoplasmic Reticulum Calcium Depletion and Disease

17. Role of Ca Signalling in Cancer Migration depends upon intracellular Ca conc. controlled by channels present at cell and organelle membrane. Ca gradient formed across cell length which increase towards rear end led to disassembly of focal adhesions (known as FAT). TRPM7 transiently allow Ca entry into cell behaving as both channels as well as kinase. Also interact with PLCb and helps in cellular migration. PIP2 hydrolysis inactivates the channel. (Runnels et al.) Normal Ca signaling is dysregulated in many cancers as TRP channels are over expressed in human breast ductal adenocarcinoma. TRPM7 is over expressed in breast and pancreatic cancer cell migration. STIM and Orai1 are important factor in breast cancer cell migration both in-vitro and in-vivo mouse models. (Yang et. al. ; 2009 - Cancer Cells)

18. Conclusion Ca and phosphate ion beside functioning as buffers, they are only ions which affects the signalling directly. Role of Ca in neurodegenerative diseases is well understood and pathway is targeted for treatment and cure. But role in cancer is yet to be discovered fully. Ca signalling is required for cell motility, maintenance and homeostasis and its role in metastasis is demonstrated in recent years. Due to ubiquitous location, this basic pathway could be targeted for cancer therapeutics.

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