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Ca2+ Signalling - Coggle Diagram

- - - - The large H+ gradient that is present drives both ATP generation and Ca2+ transport into the mitochondria
        
        Mitochondrial Ca2+ uptake complex utilises this H+ gradient to drive the transfer of Ca2+ ions
        Membrane potential controls the gating of this (it is activated once [Ca2+] = 1miM in the cytosol
        
        MCU is the pore-forming subunits of the MCUC and is regulated by MICU1&2(&3 in the brain) which confer Ca2+ regulation, which they sense through EF hands. EMRE scaffolds these two together and is required for activity of MCU units
        
        MICU1 binds to EMRE and MICU2 to MICU1 and inhibits MCU at basal [Ca2+], when [Ca2+] rises MICU2 is removed and MICU1 now interacts to stimulate MCU (both deprepression and activation)
        
        These respond to the spatial organisation of Ca2+ waves
        
        Using an aequorin fluorescent probe that is targeted to the mitochondria showed:
        1 - That local [Ca2+] reaches 10s miM
        2 - Adding a bolus of Ca2+ does not induce the same response as a histamine-induced Ca2+ spike
        
        Membrane contact site between ER and mitochondria allow for this high local [Ca2+], which triggers the MCU complex
        
        Only MCU is quick enough to oppose IP3R
        
        The response is to activate dehydrogenases and immobilise the mitochondria to ensure that enough energy is being produced and that ATP generation is close to the stimulus
        
        Immobilisation occurs as a result of Ca2+ binding Miro, which ordinarily binds to kinesin (Ca2+ binding breaks this interaction to leave the mitochondria unattached to transport machinery)
  - - - RyR(1-3) form homo-tetramers to respond to membrane depolarisation
        
        They provide the Ca2+ required for muscle contraction
        
        RyR1 is present in skeletal muscle and is localised to transverse tubules and is stimulated by a conformaitonal coupling to open L-type CaV channels
        DHPR are present in 4 per tetrad here
        
        RyR2 is present in cardiac muscle and is opened by a chemical coupling to Ca2+ ions
        DHPR appear to be randomly distributed
      - IP3R(1-3) form homo- and hetero-tetramers to respond to IP3 from PLCs
        
        IP3Rs are opened by sequential binding of IP3 (primes the channel) and then Ca2+
        
        This allows for coincidence detection where Ca2+ release activated surrounding primed channels
        
        IP3Rs are inhibited by large [Ca2+]cyt, but this has a slower response time than the activation of the IP3Rs
        Without this there would likely be explosive feedforward activation
        
        Slide 86 is the current best model for how this functions
        
        IP3 binds 7nm away from the pore and the two phosphate groups draw in the alpha and beta units (clam shell) to prime the channel (Seo, 2012, Nautre)
        
        Information is transduced through the loops in the TMDs, LNK domain is particularly important. Ca2+ binding sites are on the ARM3-LNK and ARM2-ARM1 interfaces (Fan, 2015 - well worth looking at before commenting on the regulation pathway)
        
        Highly expressed in Purkinje cells in the cerebellum
        
        Coincidence detection has been observed in these cells
        
        Each is innervated by parallel fibres and a climbing fibre, which when stimulated result in long term depression
        LTD is blocked by KO of IP3R1, mGluR1 (and restored by cell-specific expression), lack of ER in spine apparatus and inhibition of IP3-evoked Ca2+ release also inhibit LTD
        
        Two models were that there was that [IP3] was either oscillating (in line with Ca2+ oscillations) or staying stable
        
        mGluR5 has an inhibitory phosphorylation site on it, this is not present on mGluR1
        
        Using IRIS fluorescent biomarkers it was determined Matdsura (2000 or 2008) that [IP3] was in fact largely constant after an initial peak (conc increased rapidly and then oscillated at a high level before rapidly decaying)
        
        Similarly injecting a non phosphorylatable/dephosphorylatable IP3 analogue into a Xenopus oocyte triggers [Ca2+] oscillations
        
        Often done whilst trapping the IP3 analogue in a photosensitive cage and imaging with TIRF microscopy
        
        Using these biosensors has allowed imaging of blips and puffs
        
        Some IP3Rs appear to be preferentially activated
        
        Imaging by Keebler and Taylor has shown that puffs repeatedly occur at the same sites
        
        Thought to be due to the action of actin-associated KRAP, which is hypothesised to make IP3Rs competent at interacting with IP3 (licensing) - Taylor's lab hypothesises that this leads to a hierarchy of IP3Rs where only some can be primed
        
        At higher [Ca2+] the nature of the signal changes from blips -> puffs -> waves
  - - - SCID is a rare, recessive and life-threatening immunodeficiency disease caused by mutations (Arg91Trp) in Orai1 (identified through siRNA of 74 genes)
- - - - CaM has two pairs (and is transcribed from three separate genes)
        Ca2+ binding causes an unstructured region to form a helix and allow binding to another protein
    - - PLCd, PKC and synaptotagmin all have these domains to ensure that they function correctly
  - - - CaN is inhibited by Tacrolimus, which is used to prevent organ transplant rejections
    - - Forms this with various different TFs; can form a complex with AP-1 or other bZIP proteins to stimulate transcription in immune cells
    - - The same sort of oscillations can be seen by using La3+ to inhibit the Ca2+ transporters in the membrane but without Ca2+ to move across the membrane NFAT does not translocate to the nucleus
      - Spikes every 2 minutes is the optimum frequency for translocation
        
        The recovery is slow so that NFAT can accumulate in response to large and sustained Ca2+ influxes (if it was a rapid offset then no memory would be achieved)
        
        Phos/dephos cycle occurs over ~2 minutes
        IKB phosphorylates NFAT and therefore inhibits it - degradation/synthesis cycles of this occur over ~30 minutes
  - - - As a result of the masked CaM binding site, CaM only binds after CaMKII has exposed its binding site (which is more likely with a longer linker - hence longer linker = higher sensitivity)
      - The process of binding site exposure is called 'breathing' - longer linkers cause more 'breathing'
        
        The longer the linker is the lower the frequency of oscillations that is required for autonomous (high levels of) activity
    - - Phosphorylation slows CaM-Ca2+ dissociation and disables the pseudosubstrate site
      - Kinase activity is an inter-subunit interaction so neighbouring subunits must both be bound to CaM-Ca2+, hence it is very rare at low [Ca2+]
        
        Phosphorylated/autonomous subunits have an increased dissociation rate, which allows them to switch into active or inactive hubs (the latter of which then activates a given hub) (Stratton, 2014, Life)
      - Slow dephosphorylation allows for the memory of signals
- - - - PA switches off the reactions and initiates recycling
  - - - Reduced inositol results in reduced PIP and PIP2 and hence DAG and IP3, resulting in lower signalling output (inositol depletion hypothesis)
  - - - GTP-Gqa binds the C2 domain and Rac1-GTP & Gbg bind the PH domain to hold it in a specific orientation
        
        Gbg & rac1-GTP may stimulate activation by interfacial activation as the structure (Lyon, 2013) does not show movement of the pseudosubstrate
        
        Gqa binding stimulates allosteric activation by sequestering Ha2/3 that previously locked in the psuedosubstrate
    - - SH2 - Tyr-P binding results in phosphorylaiton on Y783 and this then binds cSH2 and moves the pseudosubstrate
    - - PIP2 binds the PH domain (at a non-catalytic site) and Ca2+ at the PM binds the C2 domain
        
        Doing this pushes the pseudosubstrate out of the catalytic site due to the -ve charges at the PM - this is called interfacial regulation
    - - XY linker is targeted to PI(4,5)P2 containing vesicles
      - Nesures large amount of IP3 to trigger large Ca2+ release
      - Has a different regulation mechanism with a positively charged tail
- - - - PMCA is stimulated by CaM-Ca2+ which binds at two sites (one high affinity = basal, and one low affinity = graduated response)
        
        This system allows for memory as the 1st site becomes saturated at high [Ca2+] and is then slow to dissociate
    - - Overall this is rapid for a pump though as there is little conformational change
- - - - Nir2 is important in humans (Kim, 2015, Dev Cell), the drosophila homolog is Rdg
      - The NTD binds PI/PA through a hydrophobic binding pocket (no energy input required) and the CTD binds to DAG/PA in the PM to tether it to the PM. The FFAT sequence binds VAP in the ER for a similar function