W3_L7_Calcium Signalling.pptx

PY4010 MPharm Calcium Signalling Learning objectives • To appreciated some of the properties of the calcium ion (Ca2+) that make it useful as an intracellular second messenger • To understand some of the limitations of Ca2+ ions as second messengers within the cell cytosol • To appreciated th...

PY4010 MPharm Calcium Signalling Learning objectives • To appreciated some of the properties of the calcium ion (Ca2+) that make it useful as an intracellular second messenger • To understand some of the limitations of Ca2+ ions as second messengers within the cell cytosol • To appreciated the importance and wide spread nature of Ca2+ signalling pathways in nature • To appreciated the mechanisms by which Ca2+ ions enter and exit the cell cytosol – The rising phase: voltage gated ion channels, ER/SR channels & triggers – The falling phase: Buffers, Mitochondria, Ca2+ extrusion, Ca2+ pumps, exchangers – To appreciate some important examples of cytoplasmic Ca2+ sensors (calmodulin, TnC, effectors) Ca2+ signalling….. Why Ca2+ ? The coordination chemistry of Ca2+ is such that it allows rapid, selective, reversible binding to proteins. Adapted from Carafoli & Krebs (2016), JBC 291, Ca2+ signalling….. Why Ca2+ ? Ca2+ can bind well with negatively charged oxygens (side chains of glutamate/aspartate) and uncharged oxygens (main-chain carbonyls and side chains of glutamine/asparagine). Model of Ca2+ coordination to calmodulin Aspartate Ca Glutamat e Aspartate Asparagine Ca2+ = green Oxygen atoms = red water = purple Ca2+ signalling….. Why 2+ ? Ca can co-ordinate multiple ligands (typically 7-8 but up to 12). This Ca2+ enables Ca2+ to cross link multiple segments of a protein to produce large conformational changes. Take home The coordination message: chemistry of Ca 2+ makes it highly flexible and well suited for high affinity binding to irregular shaped pockets in proteins. Extracellular vs Intracellular Ca2+ Extracellular [Ca2+]. Human body contains 1.2-1.4 kg of calcium. Over 99% of which is present as bone minerals (mainly calcium phosphate). ~1.5 g of calcium in blood (9-11 mg/100 ml, about 2.5mM).  Extracellular fluid also contains ~2.5mM Ca, about half is free Ca2+ (~1.2mM). Ca2+ as a second messenger; some considerations Extracellular [Ca2+] is high (1.2 mM) [Ca2+] in ‘intracellular Ca2+ stores’ (sarco/ endoplasmic reticulum; SR/ER) is high (1-2 mM) From Molecular Cell Biology. Eds. Londis, Baltimore, Berk, Zipursky, Matsudaira, Darnell. Scientific American Books. Resting intracellular cytosolic [Ca2+] is kept very low, 50-100 nM Ca2+ as a second messenger; some considerations Extracellular [Ca2+] is high (1.2 mM) [Ca2+] in ‘intracellular Ca2+ stores’ (sarco/ endoplasmic reticulum SR/ER) is high (1-2 mM) From Molecular Cell Biology. Eds. Londis, Baltimore, Berk, Zipursky, Matsudaira, Darnell. Scientific American Books. Resting intracellular cytosolic [Ca2+] is kept very low, 50-100 nM By controlling the Ca2+ permeability of the PM and /or the internal SR/ER membranes fast transient elevations of intracellular cytosolic [Ca2+] can be generated to drive cell Ca2+ as a second messenger; some considerations Diffusion of Ca2+ in cytosol is very slow (10-65 m2 /s, ~100x slower than that predicted for its diffusion in free solution). Barriers to diffusion arise due to the highly complex sub-cellular architecture of cells 1µm From Molecular Cell Biology. Eds. Londis, Baltimore, Berk, Zipursky, Matsudaira, Darnell. Scientific American Books. Endomembrane system Ca2+ acts as a local messenger. Why is Ca2+ signalling important/how common is it? Ca2+ signalling is the most widespread signalling pathway used in nature Mariana Leguia : https:// Why is Ca2+ signalling important/how common is it? Ca2+ signalling is one of the most widespread signalling pathways used in mammalian cells. It is also found in plants, fungi, insects, crustaceans, cephlapods, amphibians etc . How does Ca2+ enter/exit the cell cytosol? 1) Across the Plasma membrane Ion channels Ca exchangers (active) 2) From internal Ca stores Ion channels Ca pumps (active) Ca2+ transient The rising phase of Ca2+ transient Blocker s: Extracellul ar Plasma membra ne Intracellular L-type Ca2+ channel Membrane depolarisation opens channel, Ca2+ influx from extracellular matrix. Open gating mechanism resides within the aplha1 subunit Closure mechanism involves calmodulin (CaM). Ca-CaM binds with high affinity to L-type calcium channels. Targets for Ca2+ are often deep within the cell Inaccessible to Ca2+ if diffusion of from the surface membrane were the only mechanism How have cells overcome this diffusion problem?Human endothelial cell stained for PDI Store Ca2+ inside special intracellular membrane bound structures Release Ca2+ exactly where it’s needed through special ligand gated ion channels in the ER membrane PDI: Protein Disulphide 10µ M ER Ca2+ release: Inositol trisphosphate (IP3) InsP3-Ca2+ signalling: One of the most widespread signalling pathways used in mammalian cells (and plants, fungi, insects, crustaceans, cephlapods, amphibians etc) InsP3 diffuses rapidly in cytosol (~280 m2/s, ~2x slower than its diffusion in free solution). Its rate of degradation is comparatively slow (a few seconds). The net effect is that InsP3 is able to diffuse many microns in cytosol without an appreciable attenuation in its concentration. InsP3 acts as a global messenger, coupling events at the plasma membrane to intracellular Ca- release. ER Ca2+ release: Inositol trisphosphate (IP3) InsP3 is generated from the plasma membrane Inositol phospho-lipid Phosphatidylinositol (4,5)-bisphosphate (PIP2) by the action of phospholipase C Extracellul ar Intracellul ar PM G-protein-linked receptors ACh, Histamine, 5-HT, ATP PAF, Glutamate Angiotensin II, NPY, Thrombin, Endothelin etc Tyrosine-kinase-linked receptors + GTP G PLCβ InsP3 PLC  DAG PI-3K PIP3 GAP ras rat1 InsP3R Ca2+ PIP2 PGDF, EGF Antige n Ca2 ER PKC MAPK Phospholipase C family comprise four classes of enzyme those coupled via heterotrimeric G-proteins (PLC ) those that interact with activated tyrosine kinase receptors (TKR) (PLC ) those that are activated directly by high free Ca2+ (PLC ) Fertilisation Contraction Secretion metabolic changes cell motility gene expression immune cell proliferation Development Mitogenesis Ca2+ What terminates InsP3 (or transient RYR) intracellular Ca2+ release? InsP3 (and RYR) -evoked Ca2+ release is regulated by free [Ca2+]. Constant [InsP 3 ] Ca flux experiments using ER derived synaptosomes Lipid bilayer experiments measuring InsP3R channel activity Fluorescent Ca2+ indicator studies in permeabilised cells 45 At low [Ca] <300nM, InsP3-evoked Ca release is potentiated: positive feedback (regenerative) element At high free [Ca] (>1uM), Ca-release is inhibited. Bezprozvanney et al 1991 Nature 351(6329):751-4 Ca2+ transient Many cells utilise both PM and ER/SR Ca entry mechanisms Routes for Ca entry / release Tyrosine-kinase-linked receptors 7TM receptors ACh, Angiotensin II, Endothelin PGDF, EGF Antigen PLCγ PIP2 Smooth Muscle Voltagesensitive Ca channels Ligand gated channels Na+ ATP Ca2+ Ca2+ PLCβ G Depolorisati on Smooth muscle InsP3 Ca2+ - Calmoduli n (CaM) Ca2+ stores InsP3receptor Ca2+ Ca-CaM Myosin Light Chainkinase MLC MLCK MLC-P PLC = Phospholipase C InsP3= Inositol 1,4,5 trisphosphate Contraction Actin PIP2 = Phosphatidylinositol 4,5-bisphosphate Myosin light chainphosphatase SR Ca2+ release: Skeletal/Cardiac muscle Plasma membrane: Voltage gated calcium channels Dihyropyridine receptors (DHPR) SR membrane: Ligand\physicomechanical) gated calcium channels : Ryanodine receptors Morphological specialisations in large cells that need to respond rapidly Such as skeletal and cardiac muscle. Frog skeletal muscle Ryanodine receptor Type 1 (RYR1): skeletal muscle Type 2 (RYR2): Cardiac RYR1 muscle is physically coupled T-tubule (T) Sarcoplasmic reticulum (SR) to the dihyropyridine receptor (DHPR; a voltage sensitive protein…but not a functional Ca channel channel), In Cardiac muscle a functional DHPR Cardiac muscle A mixture of triggers and channels Type 2 (RYR2) Time-scale for Smooth muscle contraction elicited by different routes for Ca entry Ligand/voltage gated channels 7TM receptors PIP2 PLCβ G ~10 ms Ca2+ MLCK MLC-P Force InsP3 ~0.3 s CaCaM Ca2+ 30 ms ~1.3 s CaCaM MLCK ~250-300 ms 0.5-1.0 s MLC-P Force ~250-300 ms The time scale of cellular responses Ca2+ transient Cytosolic Calcium buffers EF-hand proteins Impact: Modulate [Ca2+]i signals Parvalbumin α Parvalbumin β CalbindinD9k CalbindinD28k Calretinin Lipids Kd for Ca2+ for most cytosolic buffers is between 0.2-2µM Resting [Ca2+]I = ~50-100nM At rest Ca2+ buffers are predominantly in their Ca2+-free state e.g. P.Serine How cytosolic buffers affect changes in [Ca2+]I depend on: 1) 2) 3) 4) The The The The buffer concentration affinity for Ca2+ (and other metal ions) kinetics of Ca2+ binding and release intracellular mobility (mobile and immobile) Ca2+ transient Mitochondrial Ca2+ buffering Human Endothelial cell histamine Cytosolic free [Ca2+] Mitochondrial free [Ca2+] Mito-DsRed GFP-proVWF How do Mitochondria take up Ca2+? MCU (Mitochondrial Calcium Uniporter) Outer Mito Membrane (fairly permeable) Allows Ca entry down its electro-chemical gradient…flux >>106 ions/sec) Inner Mito Membrane (very impermeable) Inner Mito Membrane potential difference VI---M = EM-EI = - 3Na NCX: Na-Ca exchanger (max rate 5000 ions/sec) MCU MICU1 MICU2 + Ca2+ Matrix 160mV 160mV = large driving force for Ca entry to Matrix cytoplas m MICU1/2 are MCU accessory proteins (EF-hand proteins….they bind Ca2+) MICU1/2 act as Ca sensors regulating MCU conductance At resting cytosolic Ca : MICU1 is Ca free and acts closto MCU Elevated cytosolic Ca open e : MICU1/2 bind Ca and MCU s changes in (note : outer membrane is permeable toMito Ca so these sensors can follow cytosolic ca ) Flux of Ca through MUC is far greater than that rate at which NCX operates so Matrix Ca increases (see red line in slide 28 of presentation). Matrix also contains Ca buffers that allow the Mitochondria to absorb large amounts of Ca. As cytosolic Ca declines the NXC re-sets Matrix [Ca] Mitochondrial Ca2+ buffering is important in health & disease mPTP: mitochondrial Permeability Transition Pore Mito-DsRed GFP-proVWF Hung et al (2010) Aging Research Reviews 447-456. Cardiac/lung damage Neurodegenerative Ca2+ transient Ca2+ removal from the cytoplasm Plasma membrane to extracellular space: Na+/Ca2+ exchanger Plasma membrane Ca2+ ATP-ase (PMCA). Ca2+ removal from the cytoplasm Cytosol to sarco/endoplasmic reticulum Human endothelial 10µ M SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) Ca2+ sensors Calcium signalling to effectors • Intracellular Ca2+ elevation sensors OR Ca2+ • switches – Calmodulin – ubiquitous, many functions – Effectors • Ca2+.calmodulin-dependent protein kinases • Ion channels (e.g. L-type Ca channels) – Troponin C – specific to skeletal and cardiac muscle – Effector: • Tropomyosin Calcium sensors: calmodulin (CaM) and troponin C (TnC) Cardiac and skeletal muscle Numerous targets and functions Activation of calmodulin by Ca2+ Ca2+ Ca2+-bound calmodulin embraces binding domain in target proteins via the exposed hydrophobic pockets in each lobe Calmodulin control of Nitric Oxide formation NO Influx pathway Ca2+ Nitric Oxid e Calmodulin eNos (Nitric oxide synthase PLC Phospholipase Recepto r Gprotein PLC InsP3 Ca2+ InsP3 R Ca2+ ER Ca2+ signalling dynamics Evaluation only. Created with Aspose.PowerPoint. Copyright 2004 Aspose Pty Ltd. Readin g Molecular Biology of the Cell 5th Edition Alberts, Johnson, Lewis, Raff, Roberts & Walter Advanced reading: The versatility and universality of calcium signalling (2000). Michael J. Berridge, Peter Lipp & Martin D. Bootman. Nature Reviews Molecular Cell Biology volume 1, pages11–21 Calcium signalling: dynamics, homeostasis and remodelling. Berridge MJ1, Bootman MD, Roderick HL. Nat Rev Mol Cell Biol. 2003 Jul;4(7):517-29. The Inositol Trisphosphate/Calcium Signalling Pathway in Health and Disease (2016) The rising phase • Intracellular Ca2+ stores: – Endoplasmic reticulum (ER; most cells) – Sarcoplasmic reticulum (SR; Cardiac/Skeletal Muscle) ER/SR membranes contain Ca2+ selective ligand gated ion channels Ca2+ sensors  Calmodulin (Calcium modulated protein, CaM) - Four EF-hand Ca2+ binding sites arranged in two lobes - Ca2+ binding ‘opens’ hydrophobic pockets for TnC: Ca2+ sensor for contraction in skeletal and cardiac muscle The troponin complex: TnI + TnC + TnT TnC: Ca2+ sensor for contraction in skeletal and cardiac muscle The troponin complex: TnI + TnC + TnT Activation of Ca2+/calmodulindependent protein kinases autoinhibitory/CaM binding domain Ca2+ signalling….. Why Ca2+ ? Early in evolution phosphate-based bioenergetics was selected as the energy currency of life. Calcium phosphate salts are poorly soluble. To allow phosphate-based bioenergetics, cells had to evolved processes to maintain internal calcium concentrations at very low levels. The (energy-dependent) generation of a massive Ca2+ concentration gradient across the plasma membrane was exploited to rapidly and transiently increase cytosolic Ca2+ to drive cellular processes.