Calcium Signalling

Introduction

The cellular import and export pump aid in the maintenance of calcium signaling homeostasis by altering cytosolic Ca+2 concentration, which may be reversed by pumps. Calcium is imported into the cell by voltage-dependent calcium channels (VDCC), SOCC, NSCC, NCX (exchange), and NCKX (co-transport) from the extracellular side. It may also be exported out of the ER and SR via RyR controlled by IP3R to regulate the cytosolic concentration of calcium (100nM-1mM). SERCA pumps restore calcium ions into the ER and SR after voltage-gated Ca+2 channels export calcium ions out of the cell. CaN activation (PP2B), followed by NFAT dephosphorylation. Cyclosporin and voclosporin are two medicines that target CaN.

Calcium Sensors and Adaptors

Calmodulin (CaM) - Ubiquitous adaptor protein and specifically bind to Ca+2.

❖ Contain EF Hand domain (helix-loop-helix motif) bind to Ca.

❖ Ca bind to 7 O2 atoms (chelate Ca+2) of aa in a bipyramidal pentagonal shape.

❖ Binding with Ca induces dimerization, changes in conformation, remodeling of active sites, and exposing hydrophobic surfaces.

❖ Hydrophobic surfaces wrap around target proteins, increasing CaM/Ca+2 binding specificity.

S100 and C2 Domain

❖ S100 - Ca sensory protein, also containing EF Hand domain (homo or heterodimers) helps in the assembly and disassembly of the protein complex, binding to Ca triggers hydrophobic surface exposure.

❖ C2 Domain - 120 aa stretch, contains 8 antiparallel beta-sandwich and forms a coordinate bond with Ca (bind in a cavity formed by first and final loop). Protein containing this domain is translocated to the region where Ca/CaM concentration is profound (DAG and IP3 regulated). ❖ PIP2 and CaM Switching - Both are negatively charged so they compete for positive charges created by K-Ras, ions, and EGFR. This is regulated by Ca/CaM complex, PKC phosphorylation, PIP2 hydrolysis.

Ca Pumps

❖ PMCA - removes Ca out of the cell and SERCA - restore Ca+2 inside the ER, SR making cytosolic Ca concentration.

❖ NCX - exchange 3 Na ions with 1 Ca ion and NCKX - cotransport 1 Ca with 1 K in exchange for 4 Na ions making cytosolic Ca concentration.

❖ PMCA/SERCA has a high-affinity low capacity but NCX/NCKX has a high-capacity low affinity.

❖ That means the first can retain Ca for a longer duration in the cell but the latter can make immediate adjustments in the cell.

❖ CaM regulate PMCA affinity and ATPase pump rate.

Ca Floodgate

❖ CaV conducts roughly 10 million Ca/sec, thus decreasing the 20,000-fold gradient.

❖ Channel’s antenna (helix-turn-helix) imports Calcium ions very selectively.

❖ Change in voltage (triggered by messengers) moves the paddle very quickly which opens the gate and forms a “Calcium Cage” upon binding with Ca.

Intracellular Ca Signalling

❖ Low cytosolic Ca concentration. opens the RyR channels, flowing Ca out of the ER.

❖ High cytosolic Ca concentration. block the gating.

❖ This is regulated by Ca/CaM/CaMK2, mAKAP, PKA, PR130/CaN, sorcin, and spinophilin and their defects results in cardio-arrhythmias.

TBP (Transient receptor Potential)

❖ CaV induces cell-cell by triggering protein complexes such as SNARE and synaptotagmins.

❖ IP3R releases Ca out of the ER faster as compared to Ca channeling across blood cells.

❖ 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 regulates chemokine genes on their translocation to the nucleus.

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 activate PLCb. RTK dimerizes upon ligand binding and activates PLCg.

❖ ER-mitochondrial synapse - uptake of Ca in mitochondria triggers ATP production but concentration. exceeding normal triggers apoptosis.

❖ Ca bound CaM to increase the duration of signals with the help of CaMK11 phosphorylation of substrates.

Apoptosis

❖ MiCA-mediated highly selective Ca entry in mitochondria triggers more ATP production. High or excessive ATP production and subsequent ROS production act as a defensive toxin when extruded into the cytoplasm.

❖ Microbial invasion makes releases cytochrome c from the mitochondrial outer membrane and further processes activation of caspases and Ca-dependent annexin, Bcl-2 protein translocation.

❖ Ca also regulate sperm flagellar shape but a high cytosolic Ca level could halt motility.

Smooth Muscle Contraction and Relaxation

❖ Activated Ca/CaM complex phosphorylate MLCK which phosphorylates the light chain of the myosin head and activates 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 help in releasing NOS3.

❖ NO works by activating guanylyl cyclase and PKA to lower the calcium ions levels.

❖ Cadherin and Integrin both bind to Ca and activate downstream signaling molecules such as talin, paxillin, and ⍶-actinins.

❖ N-Cadherin/b-Catenin interaction with actin cytoskeleton during contractile movement.

Role in Alzheimer’s Disease

❖ The beta-amyloid protein - neurotoxic, neurodegeneration. synaptic dysfunction and neuronal loss.

❖ Ca is an influx in high amounts through VDCC or TRP/Orai1 channel during Alzheimer's development and beta-amyloid is 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.

Role in ALS

❖ 5-10 % inherited from parents with a mutation in the SOD1 gene. SOD1 protein is localized to the outer mitochondrial membrane and involved in apoptotic signaling and oxidative stress such as mitochondrial death pathway and myocyte apoptotic signaling.

❖ The Altered Ca signaling pathway accounts for 3 interrelated toxic pathways which are mitochondrial dysfunction, oxidative stress, and neuroinflammation. (Manoj Kumar Jaiswal Djlila Mekahli et. al. - Endoplasmic Reticulum Calcium Depletion and Disease)

Role of Ca Signalling in Cancer

❖ Migration depends upon intracellular Ca concentration. controlled by channels present at the cell and organelle membrane.

❖ Ca gradient formed across cell length which increases towards the rear end led to disassembly of focal adhesions (known as FAT). TRPM7 transiently allows Ca entry into the cell behaving as both channels as well as a 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 overexpressed in human breast ductal adenocarcinoma. TRPM7 is overexpressed in breast and pancreatic cancer cell migration.

❖ STIM and Orai1 are important factors in breast cancer cell migration in both in-vitro and in-vivo mouse models. (Yang et. al.; 2009 - Cancer Cells)

Conclusion

❖ Ca and phosphate ions besides functioning as buffers, are the only ions that affect the signaling directly. ❖ The role of Ca in neurodegenerative diseases is well understood and the pathway is targeted for treatment and cure. But the role in cancer is yet to be discovered fully.

❖ Ca signaling is required for cell motility, maintenance, and homeostasis and its role in metastasis is demonstrated in recent years.

❖ Due to its ubiquitous location, this basic pathway could be targeted for cancer therapeutics.

Protein Kinase C mediated Signalling

Phospholipase C-mediated Signalling

Ca Pathway Cross Signalling