Lecture 9- Calcium signalling

Question 1

Activation of Phospholipase C

Answer 1

Phospholipase C metabolises phosphorylated lipids such as Phosphatidylinositol 4,5-bisphosphate (PIP2).

Question 2

Hydrolysis of PI(4,5)P2

Answer 2

Question 3

how Hydrolysis of PI(4,5)P2 relates to intracellular Calcium:

Answer 3

1. Activation of PLC-β by a Gαoor Gαqprotein
2. Cleavage of PIP2 into DAG andIP33.IP

3 interacts with opens Ca2+ channels on ER

4.Release of stored Ca2+ intocytosol

5. Ca2+ recruits Proteinkinase C (PKC)to themembrane
6. DAG activatesPKC
7. Active PKC phosphorylatessubstrate

Question 4

The fertilization of an egg by a sperm triggers an increase in cytosolic Ca2+

Answer 4

Question 5

GPCRs that activate or inhibit Adenylyl Cyclase

Answer 5

Adenylyl cyclase converts ATP into the secondary messenger cyclic AMP (cAMP)

Question 6

Adenylyl cyclase, cAMPand Protein Kinase A (PKA).

Answer 6

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

Question 7

An increase in cyclic AMP in response to external signal

Answer 7

In this nerve cell the neurotransmitter serotonin activates a GPCR causing a rapid increase in cAMP levels

Question 8

GPCRs that activate Adenylyl Cyclase

Answer 8

Question 9

But it doesn’t always end there…

Answer 9

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

Question 10

Enzyme coupled receptors

Answer 10

Dimerisationoccurs with ligand binding.

Proximity can cause automatic activation of a catalytic domain

or

Dimer formation may recruit associated enzymes, which then become activated

Question 11

Enzyme-coupled cell surface receptors

Answer 11

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)

Question 12

Receptor tyrosine kinase (RTK) sub-families

Answer 12

Receptor tyrosine kinases: Directly phosphorylate specific tyrosine residues on the receptor itself and on other intracellular proteins.

Question 13

Receptor tyrosine kinases (RTKs)

Answer 13

Question 14

Phosphorylated tyrosines as docking sites

Answer 14

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

Question 15

Intracellularsignallingfrom activated RTK

Answer 15

Question 16

Ras family of monomeric GTPases function as a molecular switch

Answer 16

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).

Question 17

RTKs can activate Ras

Answer 17

Question 18

Ras belong to a large superfamily of monomeric GTPases

Answer 18

Question 19

Ras family GTPases activate a MAP kinase signalling module

Answer 19

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)

Question 20

Downstream signalling pathway activated by RTKs and GPCRsoverlap

Answer 20

Question 21

Detection of Ca2+ signalling

Answer 21

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

Question 22

What methods can we use to look at this? (Ca2+ cell signalling)

Answer 22

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:

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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⁺.
7. 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.
8. Chemical Calcium Indicators
   Calcium-sensitive microelectrodes can be used to measure Ca²⁺ concentrations directly in cellular compartments or extracellular spaces.
9. 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.
10. 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