Ca2+, Lipid Derived Chemical Mediators

imissyou419's version from 2016-12-21 01:54


Question Answer
What is the concentration of FREE Ca2+ in cytosol?10^-7 M (for Ca2+ to be a good signalling molecule, must have low resting level, jump up level to interact with other proteins)
What is the concetration of Ca2+ in subcellular organelles (e.g. ER, SR, mitochondria, nuclei) or outside cell10^-3 M
Most Ca2+ binding proteins have Km ofabove 10-7 M (that is low probability of Ca2+ binding to Ca2+ binding proteins), i.e. 10-6 M
Why do we want to maintain the concentration gradient for Ca2+?High Ca2+ is toxic to cells because it can activate Ca2+ dependent proteases
Na+/Ca2+ exchanger, where it is found and what does it do, ATP?found on PM of predominantly in nerve cells, pumps Ca2+ out of cytoplasm when concentration reaches 10^-6 M locally, no ATP needed (b/c using Na+ concentration gradient)
When Ca2+ moves into the cell through Ca2+ channels at cell surface, what happens?Ca2+ comes in through electrochemical gradient AND LEADS TO LOCAL CHANGES IN [CA2+], activate Ca2+ dependent proteins and starts getting pumped out i.e. if it has 10-7M free Ca2+ inside cell, Ca2+ coming in through surface receptor does not mean all cell become 10^-3 M
Ca2+ pump, where is it and ATP?found on PM, pumps Ca2+ out of cytoplasm when concentration goes above 10^-6M, uses ATP
Ca2+ pumped into ERFunctions when Ca2+ increases to a concentration about 10^-6 M, therefore high affinity, relatively little storage capacity compared to other mechanisms, uses ATP
When Ca2+ is released from intracellular stores, what happens?Get generalized response, intracellular Ca2+ handling mechanisms to bring Ca2+ back
Ca2+ pumped into mitochondriafunctions when Ca2+ increases to high levels about 10^-5 M, therefore low-affinity, protective, relatively high storage capacity compared to other mechanisms, does NOT need ATP (energy from proton gradient in mitochondria)
Ca2+ binding to cellular proteinsCa2+ binding specifically to a number of binding proteins in cytoplasm i.e. recoverin, calmodulin, this Ca2+ is no longer "free" Ca2+ (can have large reservoir)
What are the ways Ca2+ can get into the cytoplasm to act as intracellular messenger?VG Ca2+ channels at cell surface, ligand-gated Ca2+ channels, intracellular Ca2+ stores (SR, ER) operated by IP3-gated Ca2+ channel


Question Answer
Glycerophopholipids are comprised of3-C glycerol backbone, phosphate esterified to 3rd hydroxyl - polar head group, 2 fatty acids esterified to hydroxyl groups
Fatty acid tails are usuallyheterogeneous with 14-24 C (most common is 16-18), 1 fatty acid is unsaturated (1-3 double bonds) while others are saturated -> affect membrane fluidity
Which fatty acids have no net chargePC, PE
Which fatty acids have negative net chargePS PI
Fatty acid on the middle C is oftenarachidonic acid, chopped off and made into signal mediators
PL-A1chop off fatty acid from C1, left with glycerophospholipid w/ single fatty acid left on C2 (lysophospholipid = detergent), found in snake venoms (high PL-A1 or PL-A2 activity toxic because generate lots of lysophospholipids which act as detergent and lyse membranes)
PL-A2chop off fatty acid from C2, releases arachidonic acid to make eicosanoids (prostaglandins, leukotrienes), generate lysophospholipid = detergent, regulated by Ca2+, found in snake venom (high Pl-A1 or PL-A2 activity toxic)
PL-Cdiacetyl glycerol (glycerol + lipid) retained in membrane and phospho-head group (IP3)
PL-Dphosphatidic acid = signalling molecule (glycerol + fatty acids + phosphate) retained in the membrane, release polar head group on its own (i.e. choline, ethanol)
Where do you find more PS and PI KNOW THIScytosolic side, important because PS anchors PKC, PI is cleaved to diacyl glycerol and IP3
Where do you find more glycosphingolipidsextracellular side
How is PI transformed to IP3?PI kinases uses ATP (1 more phosphate on C4 = PIP), PIP kinases uses ATP - (2 more phosphate on C4 and 5 = PIP2), happens on inner leaflet of PM
Are IP & IP2 biologically active?No
Is PIP2 biologically active?Yes if it is liberated from hydrophobic portion of the molecule (as IP3)
PIP2 concentrationsmall, produced on demand
What would happen if there was aberrant activity of PLC beta?Would not get all cleavage of PI, its substrate is PIP2 and it might not be there if there is not signalling
IP3transient, small water-soluble molecule that diffuses rapidly away from PM into cytosol, INITIATE release of Ca2+ from ER by binding to IP3-gated Ca2+ channels then get positive feedback of Ca2+
ER Ca2+ channels regulated by what feedback matter?positive feedback, Ca2+ released binds to other Ca2+ channels to release more Ca2+ in a sudden, all-or-none matter (amplification)
Is IP4 biologically active?Yes, has some biological activity (IP3 is a substrate for various enzymes - kinases and phosphatases to add and remove phosphate groups)
What are the 4 mechanisms to terminate Ca2+ responseIP3 rapidly dephosphorylated by phosphatases, Ca2+ pumping into ER (predominant), mitochondria taking up Ca2+ (back up), Ca2+ binding proteins
DAG 2 rolescleaved by PL-A2 at C2 to release arachidonic acid, activate PKC that phosphorylates selected proteins in target cells
DAG is a substrate for many enzymes and get chopped up (do not want DAG to go up every time cell signal)
PKCseveral isoforms, MOST Ca2+ dependent and ALL phospholipid dependent (depend on PS and DAG), most activated during initial rise in cytosolic Ca2+ from IP3 binding to receptor, Ca2+ leads to initial activation of PKC (causes conformational rearrangement that increases its binding affinity for DAG and PS), so PKC translocate from cytoplasm to PM where it binds PS (anchor) and is further activated by DAG (complete activation), ACTIVATED PKC IS RESTRICTED TO PM SO SUBSTRATES THAT IS PHOSPHORYLATE MUST BE LOCATED CLOSE TO PM
When Ca2+ levels drop due to Ca2+ restoring mechanisms, Ca2+ dissociate from PKC because of decreasing Ca2+ concentrations, probability of rebinding is lower, conformation of PKC changes and now has low binding affinity for DAG and PS and dissociate, cell reset itself
PKC gene transcriptionNO SIGNAL -> resting Ca2+ levels, inactive PKC, inactive MAPK, IkappaB bound to NFkappaB all out in cytosol.

SIGNAL -> ligand bind GPCR, couple Gq, Gq couple PLC beta, (PI converted to PIP2 through kinase mediated phosphorylation) PLC cleaves PIP2 into DAG (remain in membrane), and IP3, IP3 initiate Ca2+ release from ER/SR, positive feedback with Ca2+ stimulating more Ca2+ release, Ca2+ bind to PKC leading to its initial activation (conformational change so its binding for PS increases), PKC translocate to PM and binds PS (anchor), DAG leads to complete activation of PKC (at same time [Ca2+] returning to resting levels)

PKC phosphorylate MAPK and it translocate to nucleus and phosphorylate transcription factor Elk-1 (Elk-1 forms heterodimer with SRF, bind to SRE within promotor and alter gene transcription), PKC phosphorylate ikappaB which causes conformational change and now has low affinity for NF-kappaB, NF-kappaB has strong nuclear localization signal so move to nucleus and dimerize with itself as homodimer or heterodimer and regulate transcription
iKappaBmasks strong nuclear localization signal (sequences of basic and charged a.a.) of transcription factor NFkappaB


Question Answer
Proteins in the cell that are Ca2+ receptors or which are altered by increase in cytosolic Ca2+ must havehigh binding affinity for Ca2+ (binding affinity = Ka/Km ~ 10-6M) since [Ca2+] does not go beyond 6 x 10-6M generally (many proteins with Ca2+ binding motifs will have Ca2+ bound to it at 10^-6M)
Concentration of free Mg in cytoplasm, what does it tell you about Ca2+ binding proteins?10^-3 M, there is so much more Mg2+ than Ca2+, Ca2+ binding sites must be selective for Ca2+
Calmodulin found where, structurefound in all eukaryotic cells, Calmodulin can be independent protein that binds onto other proteins or constitutive element of a protein (Ca2+ binding domain within protein) to regulate their activity (target proteins include various enzymes and transporters), 2 globular motifs - Ca2+ binding sites, Calmodulin can have 4 Ca2+ bound which changes its conformation and activate the protein by binding around it, 1 flexible domain in between - able to bind around loops of protein
Allosteric binding for Ca2+/Calmodulin
when first Ca2+ binds (Km = 10^-6M), increase affinity (lowers Km = 10^-7 M), allosteric binding allows Ca2+ binding to remain high because Km gets smaller and smaller as the [Ca2+] goes down because the cell is recovering from spike in Ca2+
Does Calmodulin have any INTRINISIC BIOLOGIAL ACTIVITY?No, its biological activity is binding to other proteins and modifying activity by conformational change in that protein or be permanent regulatory subunit in enzyme
CaM-kinaseregulated by Ca2+/Calmodulin, e.g. phosphorylase kinase, myosin light chain kinase - they have very specific function and narrow substrate specificities, CaM-kinase II ((multifunctional CaMK) has broader substrate specificities
How is CaM-kinase II activated and inactivated?NO SIGNAL -> inactive CAMK II, CaMK II CONSTITUTIVELY ACTIVE, folded up in a way (COOH on top of NH2 catalytic domain) that it hides its catalytic domain; Calmodulin floating around in cell

SIGNAL -> Ligand bind GPCR, couple Gq, couple PLC beta, chop off modified GPI, IP3 released, Ca2+ goes up in cell (10^-6M), Ca2+ allosterically binds to Calmodulin, need 4 Ca2+ to activate Calmodulin, Ca2+/Calmodulin binds to Ca2+/Calmodulin binding motif on inhibitory domain of CaMK-II and PARTIALLY activate it by lifting up inhibitory domain, partially activated catalytic domain autophosphorylate itself and now is FULLY activated (catalytic domain repel)[CA2+ DEPENDENT], CaMK-II phosphorylate other substrates,
As this is happening, Ca2+ in cell returning to resting levels, 4 Ca2+ come off Calmodulin because, Calmodulin assumes different conformation so not high binding affinity for Ca2+/Calmodulin binding motif on inhibitory domain so Calmodulin comes off,

CAMK-II remain 1/2 activated (phosphorylated) even when Ca2+ levels return to normal [CA2+ INDEPENDENT](important for memory) because CaMK-II autophosphorylates itself (high negative charge so allow protein to maintain conformation where catalytic subunit is exposed to substrates in the cell & prolongs duration of activation)

Phosphatase takes off phosphate group and CAMK-II assumes inhibition but activity of phosphatase can vary so CAMK-II can be activated for a long time (hrs-days)