Lecture 9- Calcium signalling Flashcards
Activation of Phospholipase C
Phospholipase C metabolises phosphorylated lipids such as Phosphatidylinositol 4,5-bisphosphate (PIP2).
Hydrolysis of PI(4,5)P2
how Hydrolysis of PI(4,5)P2 relates to intracellular Calcium:
- Activation of PLC-β by a Gαoor Gαqprotein
- Cleavage of PIP2 into DAG andIP33.IP
3 interacts with opens Ca2+ channels on ER
4.Release of stored Ca2+ intocytosol
- Ca2+ recruits Proteinkinase C (PKC)to themembrane
- DAG activatesPKC
- Active PKC phosphorylatessubstrate
The fertilization of an egg by a sperm triggers an increase in cytosolic Ca2+
GPCRs that activate or inhibit Adenylyl Cyclase
Adenylyl cyclase converts ATP into the secondary messenger cyclic AMP (cAMP)
Adenylyl cyclase, cAMPand Protein Kinase A (PKA).
When low levels of cAMP are present, Protein Kinase A (PKA) is inactivated by binding of the regulatory subunit.
When adenylyl cyclase is activated and produces cAMP, [cAMP] increases.
PKA has greater affinity for cAMP than the regulatory subunit so cAMP binding then releases the inhibitory subunit and PKA becomes active
An increase in cyclic AMP in response to external signal
In this nerve cell the neurotransmitter serotonin activates a GPCR causing a rapid increase in cAMP levels
GPCRs that activate Adenylyl Cyclase
But it doesn’t always end there…
1.Activation of adenylyl cyclaseby a Gαsprotein
2.Conversion of ATP into cAMP
3.cAMP concentration in the cytoplasm rises
4.The regulatory subunit has greater affinity for cAMP
5.cAMP binds to regulatory (inhibitory) subunit of PKA, displacing the catalytic subunit.
6.Activated PKA can phosphorylate targets
7.Activated PKA can move into the nucleus and phosphorylate CREB, which controls transcriptional activation of gene promoter
Enzyme coupled receptors
Dimerisationoccurs with ligand binding.
Proximity can cause automatic activation of a catalytic domain
or
Dimer formation may recruit associated enzymes, which then become activated
Enzyme-coupled cell surface receptors
1)Receptor tyrosine kinases: Directly phosphorylate specific tyrosine residues on the receptor itself and
on other intracellular proteins. E.g.EGFR
2)Tyrosine-kinase-associated receptors:
Have no intrinsic enzymatic activity, but can recruit a cytoplasmic tyrosine kinase to relay the signal.
3)Receptor Serine/Threonine kinase: Directly phosphorylate specific serine or threonine residues on the receptor itself and on other intracellular proteins.
4)Histidine kinase associated receptors: Activate a two component signalling pathway.The kinase first phosphorylates itself on a histidine and then transfers the phosphate to another intracellular signal protein.
5) Receptor guanylyl cyclases: Catalyse directly the production of cGMPincytosol.
6)Receptor-like tyrosine phosphatases: Remove phosphate groups from tyrosine residues of specific intracellular signalling proteins (receptor-like because their putative ligands are not yet identified)
Receptor tyrosine kinase (RTK) sub-families
Receptor tyrosine kinases: Directly phosphorylate specific tyrosine residues on the receptor itself and on other intracellular proteins.
Receptor tyrosine kinases (RTKs)
Phosphorylated tyrosines as docking sites
The phosphorylation of tyrosine residues within the kinase domain enhances the kinase activity, and phosphorylation of tyrosine residues outside of this domain generate high-affinity docking sites for intracellular signalling molecules
Intracellularsignallingfrom activated RTK
Ras family of monomeric GTPases function as a molecular switch
Ras guanine nucleotide exchange factors (Ras-GEFs) stimulate dissociation of GDP and subsequent uptake of GTP from cytosol (activation of Ras).
Ras GTPase-activating proteins (Ras-GAPs) increase the rate of GPD hydrolysis by Ras itself (inactivation of Ras).
RTKs can activate Ras
Ras belong to a large superfamily of monomeric GTPases
Ras family GTPases activate a MAP kinase signalling module
Ras activates a cascade of mitogen activated kinases which include:
Raf –MAP kinase kinase kinase (MAPKKK)
Mek–MAP kinase kinase (MAPKK)
Erk–MAP kinase (MAPK)
Downstream signalling pathway activated by RTKs and GPCRsoverlap
Detection of Ca2+ signalling
Fura-2am
An aminopolycarboxylic acid which binds to free Ca2+
A ratiometric fluorescent dye
It is excited at 340 nm and 380 nm light
The ratio of the emissions at those wavelengths is directly related to the amount of intracellular calcium
What methods can we use to look at this? (Ca2+ cell signalling)
To study calcium (Ca²⁺) signaling in cells, various methods can be employed, each with its strengths and limitations. Here are some key techniques used to investigate Ca²⁺ signaling:
- Fluorescent Calcium Indicators
Fura-2 and Indo-1: These are synthetic dyes that change fluorescence properties in response to Ca²⁺ binding. Fura-2, for example, exhibits a shift in excitation wavelength depending on the Ca²⁺ concentration.
GECIs (Genetically Encoded Calcium Indicators): Proteins like GCaMP or Cameleon are engineered to fluoresce upon binding to Ca²⁺. These indicators allow real-time monitoring of intracellular Ca²⁺ levels with high spatial resolution. - Patch-Clamp Technique
This electrophysiological method measures the ion currents that flow through individual ion channels. It can be used to study Ca²⁺ channels’ activity and how they are regulated by signaling pathways. - Calcium Imaging
Live Cell Imaging: Using fluorescence microscopy to visualize the dynamics of Ca²⁺ in living cells over time. By loading cells with calcium indicators, researchers can visualize changes in Ca²⁺ concentration during signaling events.
Total Internal Reflection Fluorescence (TIRF) Microscopy: This method can provide high-resolution images of Ca²⁺ dynamics near the plasma membrane. - Bioluminescence Resonance Energy Transfer (BRET)
This technique utilizes bioluminescent proteins that transfer energy to Ca²⁺-sensitive fluorescent proteins. Changes in the energy transfer can indicate alterations in intracellular Ca²⁺ levels. - Calcium Flux Assays
Flow Cytometry: This method can measure changes in fluorescence intensity from calcium indicators in a large population of cells, providing quantitative data on Ca²⁺ signaling dynamics.
Plate Readers: Automated systems can measure fluorescence changes in microplate wells, allowing high-throughput screening of Ca²⁺ signaling in response to various stimuli. - Western Blotting and ELISA
These techniques can measure the expression levels of proteins involved in Ca²⁺ signaling pathways (like calmodulin or Ca²⁺ channels) or phosphorylation states of proteins regulated by Ca²⁺. - Genetic Manipulation
Using CRISPR/Cas9 or RNA interference (RNAi) to knock down or overexpress genes involved in Ca²⁺ signaling pathways. This helps determine the role of specific proteins in Ca²⁺ signaling dynamics. - Chemical Calcium Indicators
Calcium-sensitive microelectrodes can be used to measure Ca²⁺ concentrations directly in cellular compartments or extracellular spaces. - Optogenetics
This technique employs light-sensitive proteins to manipulate the activity of Ca²⁺ channels in real time. Researchers can use light to stimulate specific cells and observe the resulting Ca²⁺ signaling pathways. - Mathematical Modeling and Simulation
Computational models can simulate Ca²⁺ dynamics based on experimental data, helping to predict how cells respond to different stimuli or conditions.
Conclusion
These methods provide complementary approaches to studying Ca²⁺ signaling in cells. Fluorescent indicators and imaging techniques are particularly powerful for real-time observations, while electrophysiological methods can provide detailed information about channel activity. Combining multiple techniques often yields a more comprehensive understanding of Ca²⁺ signaling dynamics and its role in cellular processes
