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Calcium signalling Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

name 4 processes which calcium is involved in

A
  • fertilisation
  • secretion
  • gene expression
  • apoptosis
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2
Q

give three broad examples of things which can trigger a calcium response

A
  • hormones, neurotransmitter and membrane potential
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3
Q

what the differences in time period that calcium signals can last, why is this important?

A
  • calcium signals can last milliseconds or hours (fertilisation). this versatility is thought to drive the large variety of calcium dependnet processes
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4
Q

what is the concentration of calcium in the cytoplasm?

A

100nM

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5
Q

what is the concentration of calcium approximately outside the cell?

A

1mM

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6
Q

what is the concentration of calcium within intracellular stores?

A

can be 0.1-1mM- normally near the same amount that is on the outside of the cell

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7
Q

generally, how is calcium release from stores?

A
  • messenger molecules are produced in response to stimulation of specific enzymes (caused by signals) which then go on to activate calcium release from intracellular stores.
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8
Q

what are the three main calcium mobilising messenger molecules?

A

IP3, cyclic ADP-ribose, nicotinic acid adenine dinuleotide phosphate (NAADP)

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9
Q

how is IP3 created?

A

phopholipase C breaksdown PIP2 to generate IP3 which binds to the IP3 receptors of the endoplasmic reticulum

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10
Q

how is cADPR created?

A

NAD is converted by ADP-ribosyl cyclase to cADPR while also

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11
Q

how is NAADP created?

A

it is thought NADP is converted by ADP ribose cyclase

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12
Q

what is the controversy concerning how NAADP is created?

A
  • ADP ribosyl cyclase can make NAADP in a test tube but the reactions shows an acidic pH which isn’t consistent with the pH of the cytoplasm where this reaction is supposed to occur. the reaction also requires very high levels of NAADP which dont seem to be physiological
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13
Q

what is the major calcium store in cells?

A
  • the endoplasmic reticulum which is the equivalent of muscle’s sarcoplasmic reticulum
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14
Q

how are the calcium stores in the ER filled?

A

via SERCA pumps which are ATP driven

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15
Q

what is the evidence for the endoplasmic reticulum being the target of iP3? what about cADPR? what about NAADP?

A
  • you use sea urchin egg homogenates
  • you add thapsigargin which blocks the SERCS pumps. This results in a lack of calcium from the ER stores
  • then when you ass IP3 you dont get a calcium signal
  • this means that the action of IP3 is thapsigargin dependent and you know that this blocs SERCA so you know that IP3 is SERCA dependent and SERCA is on the ER.
  • the same experiment was done with cADPR
  • when you add NAADP you still get a robust calcium signal
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16
Q

how was it shown that NAADP targets calcium stores in the lysosomes?

A
  • lysosme which are used for recycling intercellular material
  • when you use GP which disturbs acidic compartments which are lysosomes, the calcium signal normally evoked via NAADP is not present
  • also when you add GPN to sea urchin homogenate you see small bursts in calcium signals which is likely the popping open of the calcium stores.
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17
Q

what receptor does IP3 work via?

A

IP3R

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18
Q

what receptor does cADPR work through?

A

ryanodine receptor on ER

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19
Q

describe the evidence which showed that NAADP, cADPR and IP3 all work via independent mechanism

A
  • if you add a signalling molecule and activate its response, you wipe of sensitivity afterwards. So you can block NAADP signalling by adding NAADP
  • when you do this and then add cADPR or IP3, cADPR and IP3 still induce a calcium signal but not NAADP
  • this also happens for the other molecules
  • this is evidence that you are dealing with different channels
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20
Q

what is thought to be the receptor for NAADP?

A

the two pore channel

21
Q

what is the structure of the two pore channel?

A
  • a duplicated domain structure which is joined with a loop structure
  • they look like half of a voltage gated channel, which has four repeats of a basic subunit
22
Q

what is the evidence that NAADP acts via two two pore channel?

A
  • TPC1 is localised to lysosomes and to endosomes
  • TPC2 is localised to lysosomes
  • if you silence the expression TPC1 you lose the calcium signal in response to NAADP
  • if you overexposes TCP you get an increase in NAADP response (in what the of cells)
23
Q

what is the controversy about TPCs being the receptor for NAADP?

A
  • a paper was published suggesting that TPCs are PI-activated sodium ion selective channels that are not responsive to NAADP
24
Q

how can calcium signals be dynamic?

A
  • calcium signals have been shown to act in waves, starting in one area of the cell and propagating: this makes them spatially and temporally complex
  • they also act via oscillations, the frequency and height of which can vary
  • they can be composed of small, spatially restricted sparks of puffs , which are thought to build large calcium signals (local vs global)
25
Q

how was calcium induced calcium release demonstrated and found?

A
  • a study measured the probability of IP3R and RyRs opening relative to the cytoplasmic calcium concentration
  • they found that the responses were bell shaped, meaning that the probability of a receptor opening is dependent on the calcium concentration: being low at low levels and high levels, with an optimal concentration
26
Q

what are the implications of calcium induced calcium responses?

A

this suggests that calcium signals can give rise to feedforward signals to increase calcium signalling

  • this can result in modest cytosolic calcium signals leading to a large calcium release
  • the descending portion of the bell curve suggests that as the concentration becomes high, the signal shuts off
27
Q

how does calcium induced calcium response link to functionality of calcium receptors and signal in terms of sparks, puffs and global signalling?

A
  • the idea is that within the ER membrane, the calcium release channels are probably clustered, and depending on the stimulus intensity, you will get different scales of channel opening
  • at low you will get opening of individual channels, responsible for small calcium blips
  • at higher you’ll get recruitment of the entire cluster called puffs which are LOCAL calcium signals
  • if you increase the stimulus intensity further you will get calcium was which cause a sweeping activity of calcium signal across the cells
  • this explains how calcium signalling can be highly localised, diffuse or global
28
Q

how can calcium induced calcium response explain calcium signal oscillations?

A

so when theyare stimulated you get a bit if Ip3 which triggers iP3 receptors then the intracellular concentration strats to rise, then because of feed forward you see an accelleration in the rate of the calcium signal- explosive- but then the calcium conc gets high and you get the descending phase of bell shaped relationship, then the channels start to shut off- terminating the calcium signal. once the channels have stopped opening, the homeostatic of mechanism kick in and brings calcium down. then relieved inhibition, allowing IP3 to open again and the process then repeats. this is an idea anyway.

29
Q

generally, what is the idea of calcium receptor chatter?

A
  • the idea is that there is crosstalk between IP3, cADPR and NAADP
30
Q

what cells were used to demonstrate chatter between calcium signalling receptors?

A

Acinar cells

31
Q

describe the experiment which first potentiated receptor crosstalk

A
  • by adding CCK you see a calcium response in acing cells
  • CCK affect works via ER so would think depends on IP3R
  • if you add NAADP, you desensitive the cells to NAADP
  • when you desensitise cells to NAADP and then add CCK, you get a very much reduced response
  • this suggests that NAADP is acting as a signalling molecule in this ER-signalling system- confusing
  • but then when you sensitive cells to cADPR agonist, you get the same effect
  • so all three molecules are interact to respond to extracellular cues…chatter
32
Q

explain the evidence for and the the model for how NAADP regulates receptor chatter?

A
  • a study looked at the effect of cytoplasmic calcium concentration of messenger-induced calcium release
  • as previously found, IP3 and cADPR signal strength was dependent on the cytosolic calcium concentration- with low causing low and very high causing low.
  • however, the found that the NAADP-induced calcium signal was not affected by the level of cytosolic calcium
  • this shows that NAADP is not able to support calcium induced calcium release and can’t undergo calcium dependent inhibition
  • this has led to the idea that NAADP acts as a trigger for calcium signalling
  • the theory:
  • NAADPR arent regulated by calcium so perhaps its role is to cause a calcium signal which then talks and provides a stimulus to activate the calcium signal which provides a stimulus to activate the more tradiitonal calcium sensiitve calcium channels
    so NAADP hijacks the calcium sensitivity of the other channels becasue it cant act this way itself
    -
33
Q

explain the evidence specifically for NAADP lysosomal signalling acting with the ER to induce a calcium signal

A
  • you induce cells to express TPC-2
  • you then see that when you add TPC2 to cells and then add NAADP that there is a calcium surge and drop signal
  • when you add Bafilomycin A1, you prevent calcium uptake into lysosomes
  • when you add NAADP after Bafilomycin A1, you see that a small calcium signal occurs- tiny.
  • this shows that NAADP TPC2 is acting via lysosomal calcium stores not ER
  • however, when you add ryanodine which blocks the RyR on the ER, and then add NAADP, you see that you also get a severe decrease in signal
  • this shows that NAADP induced signal from the lysosomal depends on the RyR action which is found on the ER
  • this shows that NAADP depends on crosstalk with ER for global signalling
34
Q

how is the trigger hypothesis at odds with the evidence from sea urchin homogenates? how can this be explained?

A

it was shown in sea-urchin homogenates, desensitisation of one receptor, didn’t affect the response to the other messengers- suggesting independent mechanisms.
- but this was done in homogenate not in an intact cell
- so in an intact cell there is some form of communication between different stores and different mechanisms. and you break this apart when you do homogenate experimentswhen you open the cell you tease apart the indivial mechanism
so this suggests that this chatter must be tighly regulated: must be very spatially regulated too.
this leads to membrane contact sites: regions of close membrane appoisition

35
Q

give two examples of membrane contact sites being significant

A
  1. one way to get calcium into the cell is through Orai proteins- they mediate calcium influx into the cell when te ER stores are depleted- so there is some communicatin between these compartments.
    Stim1 senses the depleton of calcium stores in the ER, which leads to their translocation to the contact site between the ER and the Oria calcium channels on the plasma membrane- these contact sites allow this communication
  2. in the contact of calcium signalling comes from the ER and the mitochondria.
    if you release calcium from the ER it is taken up by the mitochondira- this is because of the contact sites: and a uniporter atthe mitochondria. These contact sites bring the uniporter to the IP3R- causes a microdomian- high local calcium concnetrations which stimultaes the low affinity uniporters in the mitochondria (MICU1).
36
Q

are there contact sites between the lysosomes and ER?

A

yes!

37
Q

give an example of calcium being released at a specific region and in a wave across the entire cell

A
  • specific: secretory pole of an acinar cell

- a propagating wave: hepatocytes in the liver

38
Q

name 3 key differences between NAADP calcium signalling and cADPR and IP3 signalling

A
  1. it acts via an independent receipt and acts on the lyososmes
  2. there is a lack of Ca concentration on NAADP intracellular Ca release channel
  3. NAADP- induced calcium mobilisation is unaffected by pH, in discord with IP3 and RyR
39
Q

describe the evidence for NAADP binding being irreversible in see urchin eggs and plants and how this may affect its function

A
  • exposure to low, sub-threshold concentrations of NAADP can completely inactivate the release process to a subsequent challenge with a high concentration, despite there being no detectable Ca2+ release
  • if binding is irreversible then the receptor essentially becomes inactivated
  • if a given stimulus is sufficient to activate all NAADP receptos then NAADP calcium mobilisation may function as a one shot signal for irreversible events such as fertilisation
  • if only a small number are activated and inhibited, the magnitude of the subsequent Ca mobilisation will be decreased- this may act as a basic memory.
40
Q

give an example of how irrersible binding of NAADP in xenopus egg could be functionally relevant

A

NAADP production within a cell is non-homogeneous, possibly at the point of sperm–egg fusion, then gradients in NAADP sensitivity could result. Gradients of NAADP can be artificially generated by introducing an inactive caged precursor of NAADP into sea urchin eggs and then releasing NAADP locally in a given region by photolysis within that region only. When such a gradient is formed, the magnitude of the Ca2+ increase at a given position in the cell, in response to subsequent uniform NAADP challenge throughout the cell, is inversely related to the response following the initial local NAADP release (Fig. 1b)30. However, similar experiments with Ins(1,4,5)P3 and cADPR yield no such inhomogeneity30. Moreover, this pattern of response to NAADP persists or is ‘remembered’ for up to 20 minutes after formation of the sensitivity gradient30. This finding could indicate that, in contrast to homogenates, inactivation of NAADP receptors in the intact egg might be slowly reversible. Thus, NAADP can shape the response of a subsequent NAADP challenge, enabling cells to form a calcium memory with both spatial and temporal components, a phenomenon that could be important in events such as patterning

41
Q

is the binding of NAADP in mammalian cells reversible?

A

yes

42
Q

describe evidence in the acing cell that show chatter between NAADP and IP3/cADPR signalling

A

in the acinar cell, the response to NAADP could be blocked by either heparin or 8-amino-cADPR, antagonists of Ins(1,4,5)P3 and cADPR, respectively. There are ample data to suggest that NAADP targets a novel intracellular Ca2+-release channel.

43
Q

discuss a piece of evidence showing that NAADP acts before IP3 and cADPR during chatter

A

high inactivating concentratons of NAADP did not affect direct activation of IP3 and ryanodine receptors by their respective ligands, consistent with NAADP acting before these channels.

44
Q

describe a piece of evidence showing that there is crosstalk between cADPR and IP3 in xenopus eggs. what about evidence for redundancy?

A

Functional coupling of NAADP receptors to Ins(1,4,5)P3 and ryanodine receptors has also been demonstrated in intact sea urchin eggs. In these eggs, the propagating Ca2+ wave associated with fertilization is unaffected by blocking Ins(1,4,5)P3 or ryanodine receptors alone. However, simultaneous blockade of these receptors dramatically attenuates Ca2+ waves in response to sperm
- The potential involvement of NAADP during fertilization is supported by the finding that NAADP-induced Ca2+ release is diminished following fertilization of sea urchin eggs, presumably as a result of prior activation (and subsequent inactivation) of NAADP receptors. Thus, in contrast to data from egg homogenates, in which presumably the normal architecture of intracellular Ca2+ stores is lost, NAADP-induced Ca2+ release in the intact egg is probably a ‘summed’ response of NAADP, Ins(1,4,5)P3 and cADPR receptor activation

45
Q

discuss the study which showed that Rab acts with TPC and the effect this has on oocyte pigmentation

A
  • they expression TPCs in xenopus
  • TCP2 expression disrupted pigmentation, giving the oocyte a balding appearance
  • when done with Rab binding inhibitor, the effect was reversed.
  • overexpression of TPC2 in oocytes caused vesicular aggregation phenotype
  • NAADP-evoked response were inhibited in HEK cells expressing a Rab mutant
46
Q

how is the TPC2 overexertion on oocytes explained?

A

NAADP induced calcium released may be involved in lyososme and eadosomal fusing/ interaction- fusogenic events

47
Q

explain the link between TPCs and parkinson

A
  • because TPCs regulate trafficking events within the endolysosomal system there is the possibility that aberrant TPC activity could underlie endocytic dysfunction
    mutations in LRRK2 are a cause of autosomal dominant familial parkinson disease that is indistinyuishable from sporadic forms.
    a number of studies have implicated LRRK2 in enodlysosomal trafficiking and associated procesesses such as endocytosis and autophagy
  • they studies the endoylososmal morphology in fibroblasts from patients with LRRK2 mutation
  • they identified pronounced lyososmal morphology defects which were reversed by inhibition go TPC2
  • they also showed that using an NAADP antagonist called ned19 has the same effect
  • they reduced cytosolic levels of Ca using calcium inhibitors and found that the defects were reversed.
  • These data indicate that disrupted lysosomal morphology is likely due to dysregulated local Ca2+ signalling. This might promote Ca2+-dependent fusion of lysosomes and thus enlargement. Indeed, we often encountered large hourglass-shaped organelles delineated by a continuous membrane consistent with a fusion defect in Parkinson disease fibroblasts
  • indeed NAADP responses were significantly larger in LRRK2-PD fibroblasts than in healthy control. These signals are likely the global correlate of enhanced TPC activity that underlies the trafficking defect. Thus, both local (constitutive) and global (NAADP-regulated) Ca2+ signals are disrupted upon LRRK2 mutation.
48
Q

discuss the paper that suggests a role of TPC, NAADP in growth factor signalling

A
  • reveal an essential role for Ca2+ derived from the endo-lysosomal system in main- taining contact between endosomes and the endo- plasmic reticulum.
  • Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buff- ering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes.
  • they show that TPC1 localizes to ER-endosome contact sites and is required for their formation.
  • Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed re- ceptors with the ER-localized phosphatase PTP1B.
  • Of relevance are recent studies showing that Ca2+ regulates the formation of contacts between the ER and the plasma membrane
  • Notably, the measured affinity for Ca2+ is in the low micromolar range, sug- gesting that large global signals, such as those evoked during Ca2+ influx, regulate these junctions
  • However, because of the restricted volume at contacts, it is possible that even modest, constitutive fluxes in unstimu- lated cells, as alluded to here, could achieve the necessary Ca2+ concentrations to modulate Ca2+-dependent contacts.

Advanced molecular cell biology (6 decks)

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