Calcium Homeostasis Flashcards
Give an example of a cellular role of calcium
Concentration changes are used to transmit information through mediation of specific bond formation.
How is calcium moved through a sodium/calcium exchanger
Generally flows out of the cell down the concentration gradient (passive, no ATP required). Calcium exchanged for sodium
Where is calcium ATPase located
Endoplasmic reticulum (ER)
Where does calcium in the cytoplasm go
Pumped out of the cell by sodium/calcium exchanger
Or
Pumped into the ER (through action of calcium ATPase) or mitochondria (down electrochemical gradient)
How many calcium molecules can bind to a single calmodulin molecule?
4
What happens when calcium binds to calmodulin
Calcium/calmodulin complex formed.
Increased affinity for target enzymes due to exposure of hydrophobic regions
Inhibition of calcium/calmodulin dependent kinase 2 is stopped. Therefore it can function.
Suggest methods for measuring calcium ion levels
Radioactive labels Ion-sensitive microelectrodes Indirect electrophysiological measurements NMR spectroscopy Ion-sensitive dyes Fluorescent indicators
What types of calcium homeostasis occur in the human body
Cellular - in constant oscillation
Serum - fixed
Bone - continuously changing
What are the calcium concentrations at rest
Outside ~10^-3 M
In the ER ~ 10^-3 M
Inside <10^-7 M
Why is calcium homeostasis important
The sole function of calcium is to transmit information. Requires precise concentrations to be maintained
Summarise the movement of calcium in a cell
Passive diffusion out of the stores (mitochondria/ER) into the cytoplasm
Active transport into the ER by SERCA pumps
Diffusion into the cell through voltage- and ligand-gated ion channels
Transported out of the cell by pumps and exchangers (e.g. Na/Ca exchanger)
How is calcium pumped out of the cytosol
Na/Ca exchanger on membrane (pumps Ca out, and Na in)
Ca pump in cell membrane (pumps out calcium, but requires ATP)
How is calcium pumped into the ER and mitochondria
Ca pump in ER membrane (SERCA pump; requires ATP)
Ca-binding molecules in cytoplasm
Ca import in the mitochondria
What are the main transporters involved in calcium transportation
TRP channel (Outside calcium into cytoplasm) PMCA (calcium pumped outside) NCX (Na/Ca exchanger)(calcium pumped in/out of cell) GPCR/IP3R channel (Gq receptors activated IP3 which trigger calcium release from the ER) SERCA pump (pumps Ca into the ER)
Which calcium transporters are involved in keeping calcium concentrations low
Na/Ca exchanger BK channel (by blocking influx through VGCC/ROCC) Ca-binding molecules SERCA pump Import into the mitochondria
Which calcium transporters are involved in raising calcium levels
SMOCCs (GPCR) VGCC ROCC IP3 pathway (GPCR + ER)(activated by RTK/insulin mGluR-S etc) SOCCs
What role does Phospholipase C[beta] have with PIP3
Degrades to two messengers; DAG and IP3 (by catalysing hydrolysis of PIP3)
How does hydrolysis of PIP3 free intracellular calcium
Degraded to DAG and IP3
DAG activates PKC
IP3 activates IP3R channel which causes calcium efflux from the ER
What is the structure of IP3
6 carbon ring.
3 phosphate groups; 3 OH groups. Phosphate groups at 1,4,5 carbons.
What is the structure of the IP3 receptor (IP3R)
Tetramer. Each subunit has 6 TM domain. Each subunit has one IP3 binding site.
At least 3 known subtypes (S1-3) which modulate/express differently and found in different places
Where is the IP3R1 receptor located
Brain (esp cerebellum)
What are the two key components of calcium restocking in the ER
Sensor (to tell when the calcium store is depleted); STIM proteins
Channel (that facilitates calcium re-entry);
ORAI proteins
What is the role of STIM proteins
To detect when the ER calcium store is depleted, and activate ORAI channels
What is the structure of ORAI proteins
Tetramer protein.
3 subtypes - ORAI 1-3
What is the role of ORAI proteins
To facilitate calcium entry into the ER
When does calcium accumulation by the mitochondria occur
Commonly seen in necrotic and apoptotic cell death. May initiate apoptosis
How does calcium travel into the inner mitochondrial matrix
Outer membrane is highly permeable to calcium (lots of VDACs present).
From the outer to inner membrane calcium entry is favoured by electrochemical gradient.
What are the main pathways for calcium to get in and out of the mitochondria
Mitochondrial Calcium Uniporter (MCU)(influx) Mitochondrial Na/Ca exchanger (mNCX)(influx and efflux) Mitochondrial H/Ca exchanger (mHCX)(influx and efflux) Mitochondrial PTP (mPTP)(efflux)
What is the Mitochondrial Calcium uniporter (MCU)
Highly selective low conductance calcium channel. Allows calcium influx
What is the mitochondrial Na/Ca exchanger (mNCX)
Isoform of the NCX on the plasma membrane. Mediates low affinity calcium exchange with sodium. (influx + efflux)
What is the mitochondrial PTP (mPTP)
A channel that allows calcium efflux. Opens under pathological conditions. Also causes ATP efflux. Can activate harmful calcium-dependent proteases (e.g. calpain) to initiate cell death.
What are MAMs
Mitochondrial associated matrices
They are signalling platforms where ER and mitochondrial calcium channels interact with several modulators
How do mitochondria act as cytosolic calcium buffers
Buffering regulates calcium channel activity
Mitochondrial positioning controls calcium gradients
How does mitochondria calcium buffering regulate calcium channel activity
Rapid removal or addition of calcium modifies the local calcium concentration in the cytosol. This can open/close the calcium channel activity (think IP3). Can speed up/slow down calcium concentration oscillation
How does mitochondrial positioning control calcium gradients
Mitochondria can form a ‘belt’. They take up all the calcium, so can control the concentration that is released on the other side.
Can occur in a neuron.
How is calcium involved with IP3R activation/inactivation
Calcium promotes calcium release from IP3 receptors (self amplification)
Very high calcium concentrations can inhibit the IP3R channel and terminate release
What binding sites does the IP3R have
Binding site for IP3
Activating binding site for Calcium
Inhibiting binding site for calcium
What are the affinities for calcium at the binding sites of IP3R
Activating site has a higher affinity
Inhibiting site has a lower affinity
(therefore inhibition takes longer)
What are the names for the various channel opening events
Single - Blip
Multiple blips - Puff
Multiple puffs - Wave
How are calcium waves propagated
Calcium induced calcium release from the IP3R
What are the three main types of calcium channel
Voltage dependent calcium channels (plasma membrane) IP3-gated calcium release channels (ER membrane) Ryanodine receptor (ER membrane)
What are intercellular oscillations and how can they be used
Calcium can travel through gap junctions and signal neighbouring cells (proven with dye).
Example. Can be used in glia beta cells to synchronise calcium and release insulin.
How does the calcium/calmodulin complex activate Ca/CaM dependent kinase 2
Automatically inhibited (catalytic site blocked). Autoinhibitory domain is removed by Ca/CaM. Results in autophosphorylation and activation.
Where is Ca/CaM dependent kinase 2 mainly located
Neurones. Specifically in the postsynaptic density (PSD)
Give an example of the role played by Ca/CaM dependent kinase 2
Plays an important role in the induction of long-term potentiation (LTP). The cellular equivalent of learning and memory.
How can Ca/CaM dependent kinase 2 result in long term potentiation
When Ca/CaM is bound to the Ca/CaM dependent kinase 2, it is autophosphorylated. When Calcium and calmodulin dissociated, it remains phosphorylated (activation is prolonged).
CaMK2 can phosphorylate AMPA receptor subunits (can last ~ 30 mins), and initiate addition of new AMPA receptors
What are the downstream events associated with the NMDA receptor
Calcium increased
Calcium/calmodulin complex formed, causing:
Activation of CaMK2
Calcineurin
What is the role of calcineurin in long term depression (LTD)
Regulates phosphatase 1.
Inhibition of calcineurin blocks LTD
LTD results from removal of AMPA receptors (endocytosis)
How is dye loaded into cells to test for calcium
Acetoxymethyl esterconjugation (AM) for easy loading. Free acids (charged) loaded directly via microelectrodes
What are two methods to detect fluorescence
Conventional charge-coupled device (CCD)/camera based imaging
and
Confocal imaging
Which is the dye of choice for fluorescently labelling calcium
Fura-2 because it can be activated at two different wavelength, allowing for internal controls.
