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CORE II Block 2 > Calcium and cAMP signaling > Flashcards

Calcium and cAMP signaling Flashcards

1
Q

Why is it important to learn about calcium signaling?

A
  • Ca2+ signaling is one of the most ubiquitous processes of the cell
  • cells have hundreds of proteins evolved and adapted to specifically interact with calcium
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2
Q

List processes regulated by calcium signaling

A
  • second messenger to signal transduction
  • neurotransmitter release
  • muscle contraction
  • generation of bone
  • enzyme cofactor (e.g. blood clotting)
  • proliferation, fertilization, differentiation, synaptic plasticity, death
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3
Q

Why is calcium a useful signaling molecule?

A
  • binding to it changes protein shape and charge, which governs protein function
  • calcium imparts a positive charge, and phosphate imparts a negative charge, and both are major tools in signal transduction pathways
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4
Q

Where does the calcium used for signaling come from?

A
  • internal stores such as ER/SR
  • external medium
  • calcium always enters down a gradient
  • there is a 20,000 fold free calcium gradient (higher outside than inside the cell), and the cell expends a lot of energy to maintain this
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5
Q

Why/how is calcium carefully controlled in the cell?

A
  • it is highly reactive and can cause organelle damage or protein/nucleic acid aggregation if levels are too high
  • it cannot be chemically modified, but its concentrations are actively maintained, it is compartmentalized in the cell, and it can be chelated by BAPTA
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6
Q

Describe in general calcium binding sites to proteins.

A
  • cellular proteins bind calcium with varying affinities, which play directly to their functions
  • most common binding sites are EF hand domains (e.g. calmodulin)
  • EGF domains
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7
Q

Describe in general calcium binding to lipids.

A
  • calcium binds to the negatively charged cytosolic leaflet of the plasma membrane. This helps to rigidify and order lipid bilayers
  • calcium binding to the membrane can also modulate T cell activation, membrane fusion, and neurotransmitter release
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8
Q

Why must calcium be so strictly controlled?

A

Because it is essential for gradient formation and signaling.

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9
Q

In general, how is calcium signaling different in different cell types?

A

Each cell type expresses a unique series of receptors, channels, and other proteins that mediate calcium signaling, often generating transient calcium pulses.

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10
Q

Other than the ER/SR, what other organelles can hold intracellular calcium stores?

A

mitochondria, lysosomes, nucleus

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11
Q

How can the different functions elicited from calcium signaling be differentiated?

A

using a timescale that shows the different length pulses for different processes (e.g. exocytosis requires a short calcium pulse, and fertilization requires a much longer calcium pulse

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12
Q

List the channels that mediate calcium release into the cytoplasm from the ER.

A
  • inositol triphosphate receptor (IP3R)
  • ryanodine receptor (RYR)
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13
Q

Discuss inositol triphosphate receptors (IP3R)

A

They move Ca2+ out of the ER/SR into the cytoplasm

  • uiquitously expressed on smooth ER
  • form homo or hetero tetrameric channel into the ER
  • activation by calcium is most common calcium signaling process
  • activated by binding IP3 and low levels of calcium in the cytoplasm. binding causes conformational change which opens channel for calcium to leave the ER
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14
Q

Describe the IP3R signaling pathway.

A
  1. ligand, such as acetylcholine, binds to a receptor (GPCR or tyrosine kinase) on the plasma membrane
  2. G protein is activated and uncoupled, thus
  3. activating phospholipase C (PLC) which hydrolyzes PIP2 to form DAG and IP3
  4. IP3 and low calcium in cytoplasm go and bind to IP3R on ER membrane
  5. IP3R pore is opened and calcium is released from the ER
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15
Q

Describe ryanodine receptors (RYR)

A

Used to release calcium from ER into cytoplasm.

  • expressed in excitable cells like neurons and muscle
  • same gene family as I3PR, but has special functions
  • receptor is tetramer that opens a pore in response to activation by low Ca2+, cyclic ADP ribose, caffeine, and ryanodine
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16
Q

What major process is dependent on ryanodine receptor activation?

A

muscle contraction. The receptor opens in response to an action potential during excitation-contraction coupling, allowing calcium to flow out of the SR through RYR channels (depolarization of membrane causes spike in Ca2+ in cytoplasm, causing contraction of the muscle).

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17
Q

How do IP3R and RYR interact?

A

These two receptors do not signal in isolation. Calcium released from ER through IP3R in non-excitable cells can act as a modulatory signal in excitable cells alongside calcium released from SR through RYR to cause effects such as neuron excitation/muscle contraction.

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18
Q

List the channels used to maintain calcium gradients in the cytoplasm.

A
  • smooth endoplasmic reticular Ca2+ ATPase (SERCA) –> regulates intracellular calcium
  • plasma membrane Ca2+ ATPase (PMCA) –> regulates extracellular calcium
  • Na+/Ca2+ exchanger (NCX) –> regulates extracellular calcium
  • Na+/Ca2+-K+ exchanger (NCKX) –> regulates extracellular calcium
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19
Q

Describe SERCA and how it functions.

A

sarco/endoplasmic reticular ca2+ ATPase (SERCA) is a transporter which is the only active calcium transporter from the cytosol to the ER/SR. It moves two Ca2+ molecules into the ER for ever 1 ATP hydrolyzed.

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20
Q

Why is SERCA so critical?

A
  • calcium is constantly leaking out of the ER and needs to be put back
  • after elevated signaling using calcium, ER calcium is depleted and needs to be set up again for signaling to occur again
  • in the SR, the strength of muscle contraction is proportional to the amount of calcium released.
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21
Q

What is PMCA, and how does it function?

A

Plasma membrane calcium ATPase (PMCA) acts like SERCA, but has a different stoichiometry and moves calcium out of the cell. For every ATP hydrolyzed, 1 calcium molecule is moved out of the cytoplasm.

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22
Q

If there is a very high concentration of calcium in the cytoplasm and the cell wants to move it out, what transporter would be used and why?

A

Not PMCA, because it has a high affinity for calcium but works very slowly (good for homeostasis, not rapid removal). Instead the cell would use NCX or NCKX because they have a low affinity for calcium, but a higher capacity than PMCA.

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23
Q

How is the capacity of PMCA for calcium increased?

A

By binding to calmodulin

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24
Q

Describe NCX and NCKX. How do they differ?

A

Both channels swap intracellular calcium for extracellular potassium. They differ in their stoich.

-NCX moves 3 K+ ions into the cell

NCKX moves 4 K+ ions into the cell

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25
Q

After an action potential, which calcium transporter will be used to remove calcium from the cytosol?

A

NCX or NCKX, because there will be a lot of calcium in the cytoplasm and these transporters have a high capacity for transport.

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26
Q

What is the cellular response when calcium stores inside the cell are depleted?

A

Store-operated calcium entry (SOCE) operated by plasma membrane channels called store-operated channels. These open to allow calcium to enter the cell. These can be sustained for minutes to hours.

One oher way to replenish calcium stores is voltage-gated calcium channels.

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27
Q

Which is the prototypic store-operated channel (SOC)?

A

calcium release activated calcium (CRAC) channels.

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28
Q

As ER calcium levels decrease, what happens to CRAC activity levels?

A

They increase.

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29
Q

What is the structure of CRAC channels? How do the components function?

A
  • made up of the ORAI channel which is in the plasma membrane (four transmembrane domains that interact with STIM)
  • STIM (1 and 2) channels in the ER membrane which bind calcium in the lumen. When unbound during low calcium in the ER, the STIM channels dimerize, forming a channel through which calcium can enter. When bound by calcium in the lumen, the dimer undimerizes, breaking the channel.
30
Q

To what domains on STIM 1 and 2 does calcium bind?

A

To EF hand domains on the STIM proteins, allowing them to be calcium sensors.

31
Q

How can the formation of CRAC channels be visualized?

A
  • STIM can be stained with mCherry
  • ORAI can be stained with GFP

when stores are full, we can see these channels dispersed throughout the membranes. When calcium stores in the ER are depleted, we can see the channels overlapping, lining up as a CRAC (type of SOC channel)

32
Q

Describe voltage gated calcium channels (VGCC) and their role in calcium homeostasis.

A

They help move calcium into the cell when ER/SR stores are depleted. These are involved in action potentials, and are therefore found in excitable cells like neurons and muscle. It is a multimeric complex of different subunits, where the a1 subunit forms the ion channel.

33
Q

List the mitochondrial calcium channels

A
  • VDAC
  • MICU
  • NCLX
34
Q

Describe VDAC channels.

A

Voltage-dependent anion-selective channels (VDAC) are abundant in the mito outer membrane allow ions (not just calcium), nucleotides, and other metabolites into the mito.

  • conformation is voltage-dependent (in the name)
  • closed conformation is more selective for calcium
  • interacts with ER to enhance IP3-induced calcium signaling
35
Q

Describe the mitochondrial calcium uniporter (MCU) and how it functions.

A
  • MCU pore in inner mitochondrial membrane, regulated by MICU1 and MICU2 (heterodimer)
  • when calcium signaling begins and cytosolic calcium levels increase, some calcium diffuses into inner membrane space, binds the MICU heterodimer, and opens the MCU pore so calcium can enter the mito matrix.
36
Q

How does calcium exit the mitochondria?

A

In excitable cells, the sodium-lithium-calcium exchanger (NCLX) moves calcium out of the mito matrix in exchange for lithium or sodium

37
Q

What defect causes mitochondrial calcium overload? What pathology results?

A

Mito calcium overload is caused by the deletion of the NCLX, and causes cardiac necrosis and death from heart failure.

38
Q

List the lysosomal calcium channels.

A
  • two pore segment channel 1 (TPC1)
  • transient receptor potential cation channel, mucolipin subfamily (TRPML)
39
Q

What causes different calcium signals to have different lengths and to reach different amounts of space?

A

This depends on the size and number of targets of the calcium signal

40
Q

List the different types of calcium signals.

A
  1. fundamental calcium release (blip/quark aka single channel activation)
  2. elementary calcium release (puff/spark, several channel activation)
  3. global calcium release (wave, many channels opening in tandem)
41
Q

Difference between puff and spark? Similarity?

A

Difference

  • puffs are more diffuse and last longer
  • sparks are more localized and shorter-lived

Similarity

-cooperative activity between RYR and IP3R on ER surface at a single release site

42
Q

Describe calcium waves.

A
  • elevations in calcium involving multiple release sites (domino effect)
  • can be within a cell or among cells
  • begins with increase in cytosolic calcium followed by a succession of similar events
43
Q

How might a calcium wave begin?

A

Perhaps by an action potential (intercellular) or during fertilization (intracellular)

44
Q

Why does calcium pass through a channel quickly, but slow down once it reaches the cytoplasm?

A

calcium begins interacting with sensors and binders such as calmodulin, which slows down its diffusion. This can result in non-homogenous activation of calcium binding proteins

45
Q

What is the role of calcium sensor proteins?

A
  • calcium sensors like calmodulin are bound by calcium and can sense different concentrations, such as during a signal. These relay info by binding to target proteins.
  • Calcium dependent and independen kinases relay signals by phosphorylating target proteins
46
Q

Describe calmodulin and calmodulin activity.

A
  • small calcium sensor/adaptor protein
  • highly conserved with 4 EF hand domains, each lobe having different affinities for calcium
  • binding of calcium reshapes the lobes and exposes hydrophobic surfaces, enabling binding to target proteins
  • binding to target proteins can cause relief of autoinhibition, remodeling of target protein active sites, and protein dimerization
47
Q

Describe the role of calcium signaling in immune function.

A
  • in T cells, MHC protein presents something to the T-cell receptor, causing calcium to be released from the ER
  • CRAC channels are then activated to restore the ER stores, while the cytoskeleton maintains high levels of calcium in the cyotplasm.
  • the maintained cytosolic calcium levels mediate a varierty of T-cell functions
48
Q

What is the role of calcium signaling in neurological disorders?

A

Neurological disorders often have altered calcium homeostasis

49
Q

How can calcium in cells, cell populations, and within tissues be imaged?

A

calcium indicators can be used to image calcium. These are fluorescent molecules that respond to being bound by calcium by changing their fluorescence. Once the molecule has been added to the cell(s), they are then exposed to the molecule or process thought to increase calcium while set up under the microscope.

50
Q

How does a researcher decide what type of calcium indicator to use for imaging calcium in a cell or tissue?

A
  • length/magnitude of calcium release
  • experience of the researcher
  • modality (microscope) being used
51
Q

What are the types of chemical indicators for calcium signaling?

A
  • ratiometric (fluorescence at 2 wavelengths) ex: fura-2
  • single wavelength (fluorescence at 1 wavelength) ex: rhod-2
52
Q

Why are chemical indicators for calcium signaling modified?

A

they are charged and can therefore not pass the plasma membrane as is. therefore, they are esterified to acetoxymethyl in order to pass the membrane. Once through, an esterase removes the AM, restoring the indicator’s function.

53
Q

Which type of chemical indicator is good for an unexperienced researcher to image calcium?

A

fura-2 AM

54
Q

What are genetic indicators? How can they be used to image calcium in the cell?

A

They are often GFP fused with the calcium-binding sites on calmodulin. FRET is then performed. When calcium is not bound, fluorescence is low. When calcium binds, there is a structural shift indicated by an increase in fluorescence. The difference to chemical indicators is that the indicator is already inside the cell and does not need to be loaded separately.

55
Q

How is cAMP signaling stopped?

A

Degradation by phosphodiesterases. This is how cAMP signaling can be controlled. PDEs hydrolyze the 3’ cyclic phosphate bond of cAMP.

56
Q

What does cAMP have in common with calcium, IP3, and diacylglycerol?

A

They are both intracelular second messengers

57
Q

What is the overall activity of a second messenger?

A
  1. agonist activates membrane-bound receptor
  2. G-protein is activated and produces effector
  3. effector stimulates second messenger synthesis
  4. second messenger activates intracellular process
58
Q

How is cAMP made?

A
  • catalyzed by adenylyl cyclase (AC). Both plasma-bound and cyotplasm-resident isoforms with highly conserved catalytic domains.
  • AC is activated by the Gas subunit of G-protein
59
Q

How does adenylyl cyclase mediate activation of PKA?

A

by catalyzing the synthesis of cAMP which activated PKA

60
Q

Which G-protein subunit binds guanine?

A

Ga

61
Q

How is the GDP molecule bound to Ga exchanged for GTP to activate G protein trimer dissociation?

A

Guanine exchange factor exchanges GDP for GTP.

62
Q

What different effects do the different Ga subunits have on AC?

A
  • Gas stimulates AC, making more cAMP in the cell
  • Gai inhibits AC, so the cell has less cAMP
63
Q

Describe the activity of PKA.

A

Protein kinase A is dependent on activation by cAMP. It is a heterotetrameric holoenzyme with a regulatory dimer and 2 catalytic subunits. After cAMP binding to regulatory dimer, c-dimer dissociates and can act as an effector.

64
Q

What types of processes do PKA regulate?

A

Activated PKA is involved in transcriptional regulation and activty of other enzymes.

65
Q

How is the location of PKA signaling in the cell regulated?

A

A-kinase anchoring proteins (AKAP) anchor PKA through a docking domain on the regulatory dimer of PKA. The AKAP-PKA complex is directed to discrete cellular locations which affect the activity of PKA

66
Q

What type of pharmaceutical drug uses PDEs?

A

erectile dysfunction drugs

67
Q

How can cAMP pathways be monitored in the lab?

A

Can do ELISA, fluorescence, etc. What is most importance is to inhibit PDEs so that the cAMP being studied is not degraded. To study downstream activity of cAMP, often CREB is examined (binds DNA. also used to measure PKA activity indirectly)

68
Q

How can calcium regulate adenylyl cyclase?

A

AC1, AC8 activity increased by calmodulin

AC5, AC6 activity inhibited by calmodulin

AC9 regulated by calcineurin

69
Q

How does calcium regulate cAMP levels?

A

Calcium regulates different phosphodiesters and therefore cAMP levels and signaling

70
Q

How can cAMP regulate calcium channels? Specific example?

A
  • in heart: adreneline activates receptors bound to G-protein, activating cAMP and PKA
  • PKA phosphorylates L-type calcium channels, allowing an influx of calcium from outside to inside the cell (associated with contraction)
  • phosphatases stop this signal by removing phosphate from the calcium channel

CORE II Block 2 (6 decks)

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