Lecture 11 12 13 MCQ PDF

Calcium as a second messenger BC 3004 / BC3021 Cell signalling Lecture topic: Ca2+ signaling 1  Calcium as a second messenger (Part I) Effects of intracellular Ca2+ on cellular processes: § Many hormones as well as other extracellular st...

Calcium as a second messenger BC 3004 / BC3021 Cell signalling Lecture topic: Ca2+ signaling 1  Calcium as a second messenger (Part I) Effects of intracellular Ca2+ on cellular processes: § Many hormones as well as other extracellular stimuli (neurotransmitters) elicit increases in free cellular Ca2+ leading to diverse cellular responses: EXOCYTOSIS. e.g.neurotransmitter MUSCLE release CONTRACTION REGULATION OF Ca2+-DEPENDENT CONTROL OF ION CHANNELS --> METABOLISM MEMBRANE EXCITABILITY CONTROL OF Ca2+ REGULATION OF Ca2+-DEPENDENT CELL-CYCLE/ KINASES AND SURVIVAL PHOSPHATASES DIFFERENTIATION ACTIVATION OF REGULATION OF Ca2+-DEPENDENT GENE EXPRESSION PROTEASES AND AND PROTEIN NUCLEASES SYNTHESIS REGULATION OF 2 FERTILISATION  The concentration of intracellular free Ca2+ is controlled by its uptake and release from membrane limited cellular compartments Two main calcium stores = ER (SR in muscle), mitochondria Extracellular Ca2+ Ca2+ Ca2+ 1-2mM Ca2+ Mitochondrion Ca2+ Active Resting µM Ca2+ ~100nM Ca2+ ER Ca2+ (10-100µM) Ca2+ channel Ca2+ Ca2+ Ca2+ ATPase 3  Intracellular free Ca2+ can be rapidly increased by two mechanisms 1) Influx of Ca2+ ions from the EXTRACELLULAR environment, via ion channels: a) Voltage-operated Ca2+ channels (VOCCs): open in response to membrane depolarisation, e.g. the dihydropyridine receptor (also called voltage- dependent calcium channels) b) Receptor-operated Ca2+ channels (ROCCs): these are directly coupled to receptors at the cell-surface, e.g. the ionotropic glutamate receptors can conduct Ca2+ ions 4  Intracellular free Ca2+ can be rapidly increased by two mechanisms c) 2nd messenger-operated channels (SMOCCs): open in response to increases in the cytosolic concentration of second messenger species, e.g. IP4 receptors, cGMP-gated ion channels d) Mechanically-operated Ca2+ channels (MOCCs): are gated in response to mechanical stimulation of the cell, e.g. shear stress 2) Release of Ca2+ ions from INTRACELLULAR stores: endoplasmic/sarcoplasmic reticulum (ER/SR) in most cells. Two different families of channel protein are involved in Ca2+ release: 5 a) IP3 receptors are: i. Located on the ER ii. Expressed ubiquitously with greatest abundance in the brain (NT release) iii. Activated by the 2nd messenger IP3 to release Ca2+ from the ER lumen iv. Tetrameric complexes of four high MW (~310 kDa) subunits v. At least three distinct isoforms of IP3R protein exists in mammals…… b) Ryanodine receptors (RyRs) are: i. Located on the SR of heart muscle, skeletal muscle and the nervous system ii. Sensitive to the plant alkaloid ryanodine iii. Tetrameric in structure (~560 kDa) iv. There are at least 3 RyR isoforms in mammals 6  Opening IP3R and RyR channels displays a dependency on cytosolic Ca2+ concentration: § Called Calcium induced calcium release (CICR) § Threshold concentration of free calcium is required for complete opening of the channels § Acts as an amplification mechanism § BUT once cytosolic Ca2+ is increased beyond a certain point……inhibits further opening of the channels = negative feedback § Different IP3R and RyR isoforms show quantitatively distinct dependencies on cytosolic Ca2+, e.g. type 1 RyR shows maximal activity at about 10 µM Ca2+, whereas the type 2 is most active at about 1 µM Ca 7 2+  IP3Rs require IP3 to be activated: § Ca2+ acts as a co-activator (CICR) RyRs can be directly activated by Ca2+ (CICR), or by mechanical interaction with dihydropyridine receptors, which act as voltage-sensors in the plasma membrane, responding to membrane depolarisation: 8  Removal of calcium from the cytosol: 1) Ca2+ ATPases in the PM and on the ER/SR and mitochondria 2) Association of Ca2+ with binding proteins which buffer it e.g. calmodulin, calsequestrin and calreticulin 3) Ca2+ can also be bound to negatively charged phospholipids (µM range) Measurement of intracellular calcium: § Calcium binding fluorescent dyes e.g. fluo-3, fura-2 § Calcium sensing proteins e.g. aequorin § 45Ca2+, radiolabelled Ca2+ 9 Intracellular calcium as monitored with Fura-2 10  Malignant Hyperthermia Inherited disorder of skeletal muscle Affects humans, pigs, horses Hypermetabolic response to inhalation of anaesthetics such as halothane, succinylcholine Increased O2 consumption and CO2 production High increase in body temperature, tachycardia……skeletal muscle rigidity (paralysis), organ failure Can occur in minutes or hours 11  Malignant Hyperthermia MH gene: RyR1 gene on chromosome 19 (humans). Many different mutations (up to 18)……uncontrolled release of Ca2+ from the SR in muscle (50 - 70% of cases). Autosomal dominant. Prevalence of MH is unknown MH has also been linked to other loci……chr. 1, 3, 7, 17 Diagnosis……at present muscle biopsy…… caffeine/ halothane……muscle contraction Treatment: Dantrolene: muscle relaxant 12  Calcium as a second/third messenger (Part II) Many Proteins act as effectors for Ca2+ signals either directly or indirectly: 1) Ca2+- binding proteins, whose structure/function changes on binding this ion e.g. calmodulin, calreticulin 2) Effectors whose activity is modulated indirectly by Ca2+, via Ca2+- binding proteins 13  Calmodulin Calmodulin is a very important protein which binds calcium and in turn regulates the structure and function of several other proteins Note: EF-hand and C2 domains are capable of binding Ca2+ Calmodulin: § 17 kDa calcium binding protein § Ubiquitously expressed i.e. present in most cells in eukaryotes § Four Ca2+-binding sites, termed EF-hands , arranged in pairs at either end of the protein 14  Calmodulin On binding Ca2+, calmodulin undergoes a large conformational change, from a dumbbell structure, to a more compact, spherical form, wrapped around a target protein Ca2+/calmodulin bound to Calmodulin without calcium target peptide 15  Calmodulin Target proteins: § Protein kinases (calmodulin-dependent protein kinase II, myosin light chain kinase) § Protein phosphatases (calcineurin) § Other enzymes (glycogen phosphorylase kinase) § Transporters (Ca2+ ATPase) § Ion channels (RyR, IP3R) 16  Calmodulin-dependent protein kinase II (CaMKII) A key cellular effector Activity depends on calmodulin and calcium ions Serine/threonine kinase Ubiquituous tissue distribution Multifunctional kinase 5 isoforms: M.W. 50-60 kDa. Active CaMKII: 600kDa oligomer Soluble cytosolic protein 17  Calmodulin-dependant protein kinase II (CaMKII) A multifunctional kinase……phosphorylates numerous effector proteins thus influencing a number of cellular processes like: § Stimulation of neurotransmitter release…… phosphorylates synapsin 1 § ……effects on membrane trafficking machinery (SNAREs, Rabs, ARF proteins) § Activation of neurotransmitter and hormone synthesis e.g., CaMKII phosphorylates and stimulates tyrosine and tryptophan hydroxylases, which are involved in the synthesis of the catecholamines and serotonin, respectively 18 Calmodulin-dependant protein kinase II (CaMKII) § Stimulation of calcium sequestration in heart muscle (phosphorylation of phospholamban in heart muscle, a protein which regulates the activity of Ca ATPase pumps involved in the uptake of this ion into the SR) § Regulation of transcription through phosphorylation of several transcription factors § Misregulation of CaMKII have been linked to Alzheimer’s disease, Angelman’s syndrome, and heart arryhthmia. 19  Tryptophan hydroxylase: § stimulation of serotonin biosynthesis Tyrosine hydroxylase: § stimulation of catecholamine biosynthesis Synapsin I: § NT release Glycogen synthase: § Suppression of glycogen synthesis RyR, IP3R s, PM Ca2+ATPase = targets of CaMKII 20  Calcineurin (protein phosphatase 2B) Ubiquituous serine/threonine protein phosphatase Ca2+ dependent. Calmodulin acts as a cofactor for activity 2 subunits: 1) A subunit = catalytic (61 kDa) 2) B subunit = regulatory (19kDa) Related to calmodulin Substrate specific: Targets = PKA Thus involved in cross-talk between cAMP-coupled receptors and Ca2+ second messenger systems 21  View the following you tube clips Calcium as a second messenger (4 mins long) https://www.youtube.com/watch?v=v-hHIACJ9SE Second messengers: cAMP, cGMP, IP3, DAG & Calcium (13 mins long) https://www.youtube.com/watch?v=PzA5Z3DXfrQ 22  Protein kinase C Serine/threonine protein kinase……activated by DAG and Ca2+ In mammals several isoforms exist (at least 6 isoenzymes) It is cytosolic but can translocate to PM in presence of DAG ( docking factor )……phosphorylate membrane- associated proteins Ubiquituous - but enriched in brain Functions as (70kDa) monomer in cell proliferation, differentiation, angiogenesis and apoptosis C-terminal catalytic domain and an N-terminal regulatory domain (binds DAG, calcium) Can be activated by phorbol esters e.g. phorbol myristyl 23 acetate (PMA) that mimic DAG……tumour promoters Protein kinase C 24 Signalling Lipids – The Eicosanoids BC 3004 /BC3021 Cell signalling Lecture topic: Eicosanoids 1  View you tube clip on the Eicosanoids https://www.youtube.com/watch?v=FxZsd-heeQA 2  EICOSANOIDS AND OTHER LIPOPHILIC MOLECULES WHICH BIND CELL-SURFACE RECEPTORS The eicosanoids can be divided into four subfamilies, the prostaglandins, prostacyclin, thromboxanes, and leukotrienes. Eicosanoids are 20-carbon fatty acid derivatives which are synthesised from polyunsaturated fatty acids, particularly, though not exclusively arachidonic acid. The name eicosanoid derives from the Greek ‘eicosa’, meaning ‘Twenty’. Arachidonic acid has a 20 C: 4 unsaturated bond structure 3  Properties of Eicosanoids The eicosanoids are poorly soluble in water. They are produced and act locally, as autocrine or paracrine local mediators. Despite their hydrophobic chemistry, they bind to receptors on the cell-surface. Eicosanoid receptors typically are seven transmembrane receptors and signalling occurs via heterotrimeric G- proteins i.e. GPCRs The eicosanoids have a very wide range of biological functions, which include inflammation, pain sensation, platelet aggregation, asthma. 4  Properties of Eicosanoids There are four main classes of Eicosanoids: Prostaglandins (PGE2, PGD2, PGF2a) Prostacyclin (PGI2) Thromboxanes (TxA2) Leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4) Most cells produce at least a few types of eicosanoid. Prostaglandins were originally identified in the prostate gland, hence explaining the derivation of their name. Subsequently, it was realised that most cells in the body can produce these lipid signalling molecules. All known types exert their effects on target cells by binding to cell-surface (7 TM) receptors coupled to second messenger systems. 5  Eicosanoid Biosynthesis Eicosanoids are not stored in significant amounts in signalling cells. Synthesised when a cell is activated by mechanical trauma, growth factors, cytokines, or other stimuli. Their rate of release is determined mainly by the rate of synthesis, which in turn is largely dependent on the availability of their precursor (predominantly arachidonic acid). 6  Eicosanoid Biosynthesis The precursor, arachidonic acid, an essential fatty acid, cannot be synthesised de novo (from scratch) in mammalian cells. It is thus said to be an ‘essential’ fatty acid. Arachidonic acid (AA) is normally present in cells esterified at the 2-position in phospholipids, where it can be released by the action of phospholipase A2 enzymes. It can also be produced by the elongation and desaturation of other essential fatty acids, e.g. linoleic acid. 7  Eicosanoid Biosynthesis At the Endoplasmic Reticulum (ER), and nuclear membranes, arachidonic acid released by PLA2 is presented to prostaglandin H synthase (PGHS - more commonly known as COX - the acronym for cyclooxygenase). PGHS/COX converts AA to prostaglandin H2 (PGH2) Enzymatically catalysed reduction or isomerisation of prostaglandin H2, generates a range of eicosanoids i.e. different classes prostaglandins……prostaglandin E2, A2, also prostacyclin, thromboxane A2 8 Eicosanoid Biosynthesis 9  Eicosanoid Biosynthesis Regulation of PLA2 is incompletely understood but it involves phosphorylation and an influx of Ca2+ which triggers PLA2 activation and translocation to the nuclear membrane. Phosphorylation of PLA2 at serine-505 is thought to be the result of ligand binding to receptors such as the interferon receptors, mGLUR1, etc. Glucocorticoids stimulate the release of lipocortin which inhibits PLA2 and reduces inflammation. 10  Eicosanoid Biosynthesis Note re. synthesis of Arachidonic acid (AA): AA can also be generated by the action of phospholipase C (PLC) on phospholipids to form diacylglycerol (DAG). This can then be cleaved by diacylglycerol lipase to form arachidonic acid and monoacylglycerol. *For more details on the production of the prostaglandins, prostacyclin and thromboxanes by the actions of cyclo-oxygenase enzymes (COX) on polyunsaturated fatty acids (usually arachidonic acid) see Fig. 3.8 in Eicosanoid HO pdf. 11 Eicosanoid Biosynthesis 12  Eicosanoid Biosynthesis Two isoforms of Prostaglandin H synthase are known to exist: PGHS-1 (COX-1) and PGHS-2 (COX-2); COX-1 is responsible for basal/constitutive prostaglandin synthesis COX-2 is important for induced prostaglandin synthesis such as those involved in the inflammatory response. 13  Inhibition of Eicosanoid Synthesis Several Therapeutic Drugs Target Eicosanoid signalling A major group of anti-inflammatory/pain killer drugs mechanistically target eicosanoid biosynthesis – the non- steroidal anti-inflammatory drugs (NSAIDs). Classical NSAIDs include; Aspirin - used to control blood clotting & pain killer Ibuprofen - an anti-inflammatory painkiller The NSAIDs target COX-1 and COX-2 to varying extents. Therapeutically, the preference is to target COX-2 more 14 effectively.  Cyclooxygenase Inhibition Cyclooxygenases are inhibited by aspirin and ibuprofen. Thereby prevent the production of eicosanoids. Aspirin = pain killer………..eicosanoids are important in pain perception. Aspirin also has anti-coagulant properties, by inhibiting the formation of thromboxane A2 e.g. low doses of aspirin are commonly used in the prevention of stroke. Thromboxane A2 is released by platelets and causes platelet aggregation, which leads to blood clotting. 15  Cyclooxygenase Inhibition Aspirin has a number of side-effects including gastrointestinal bleeding and has been linked to Reye’s syndrome. Contraindications are linked to its inhibition of COX-1. Therefore pharmaceutical companies developed COX-2 specific inhibitors (coxibs). 16 Cyclooxygenase Inhibition 17  Regulation of Eicosanoid Signalling Production of eicosanoids is often regulated by the action of other hormones or stimuli on signalling cells e.g. mechanical trauma, cytokines, growth factors or inflammatory stimuli. Eicosanoids act as ‘local mediators’ (autocrine + paracrine signalling), since they are usually degraded by extracellular enzymes before they can interact with distant target cells. They often act in ‘antagonistic pairs’. For example, thromboxane A2 causes platelet aggregation, but prostacyclin, which is released from the vascular endothelial cells (lining the blood vessel), has the opposite effect. This interplay controls local blood clotting. 18 Regulation of Eicosanoid Signalling 19 20  Leukotrienes Unlike the other eicosanoids, leukotrienes, are not synthesised by the action of cyclo-oxygenases. 1. They are synthesised by lipo-oxygenases 2. Oxidise the 5-carbon of arachidonic acid to form a hydroperoxy fatty acid intermediate. 3. Several of the leukotrienes are modified by conjugation to more polar molecules, such as glutathione (leukotriene C4) or cysteinylglycine (leukotriene D4). 21 Leukotriene Biosynthesis *see Fig 3.9 of Eicosanoid HO pdf for structures 22  Leukotrienes Leukotrienes cause the contraction of airway smooth muscle (LTD4) and are believed to play a role in the etiology of asthma. Leukotrienes also cause neutrophil chemotaxis (LTB4) and oedema. 23  Other lipophilic hormones that bind cell surface receptors Examples include platelet activating factor (PAF) and lysophosphatidic acid (LPA). They are structurally distinct from eicosanoids. These hormones can act over longer distances than eicosanoids……transported in the blood. 24  Other lipophilic hormones that bind cell surface receptors Platelet activating factor triggers platelet aggregation. Lysophosphatidic acid (LPA) has a number of effects on targets, including - stimulation of growth, cell-division, transformation and alteration of cell morphology. Both of these ligands bind to distinct families of receptor, coupled to intracellular signalling pathways. 25 G-proteins in sensory perception BC 3004 /BC3021 Lecture topic: Vision/Smell/Taste 1  G proteins in sensory perception Vision/Smell/Taste: Broad overview, with a focus on vision and olfactory signal transduction mechanisms 2 Vision 3  G proteins in sensory perception Vision: Eye Light photon retina Rods Cones 3 types: Black & white vision Colour vision Blue Vision at low light levels Green Red light Optic nerve 4 G proteins in sensory perception Organisation of cells in the retina 5 Rod cells have a characteristic structure 6 Figure 15-16  Rod cells have a characteristic structure The membrane discs within the outer segments of these cells are highly enriched in light-sensing proteins known as rhodopsins Rhodopsins are G-protein coupled receptors which are activated by photons of light Rhodopsins: chromophore called retinal (vitamin A derivative) covalently linked to a protein, opsin Both rods and cones use retinal to sense light but the retinal is complexed to different isoforms of opsin……thus different rhodopsins……have distinct light absorbance spectra 7  Rhodopsins 7-transmembrane receptors (7-TMRs) Coupled to heterotrimeric G-protein - called transducin Transducin undergoes GDP-GTP exchange……activation Stimulates phosphodiesterase which cleaves cGMP to GMP Normally cGMP stimulates opening of Na+ channels…… depolarisation = nerve impulse Mechanism of action: Upon receiving a light photon rhodopsin activates transducin to stimulate cGMP cleavage by phosphodiesterase GTPase activity of transducin causes self- 8 inactivation……GTP is hydrolysed to GDP  Rhodopsins Mechanism of action: Also calcium ions can normally enter the cell from extracellular fluid through Na+ channels. Low cGMP = Na+ channels close……decrease in intracellular calcium…… activates guanylyl cyclase enzymes to increase cGMP……opening of Na+ channels All of this allows photoreceptor cells to switch off rapidly after a flash of light…allows the measurement of the intensity and duration of the flash……vision 9  Visual Phototransduction Pathway Plasma membrane Outer membrane disk Step 1: A photon of light activates rhodopsin (R*) by inducing a conformational change. Step 2: R* activates several transducin G-proteins by exchanging GDP for GTP (G*). Step 3: G* activates phosphodiesterase E. Step 4: Activated PDE hydrolyses cGMP to GMP. This causes cGMP-gated ion channels to close preventing influx of Na+ and Ca2+. Step 5: Reduced cGMP levels trigger guanylyl cyclase to synthesise more cGMP and leads 10 to opening of cGMP-gated ion channels. Smell 11  Odours and Odorant Receptors Humans can detect greater than 10,000 smells (odorants) Do so via olfactory receptors……GPCRs (~ 1,000 in humans) Distinct olfactory receptors are expressed on specialised cilia of distinct olfactory receptor neurons (ORNs) within the nose They transduce a chemical signal (odorant) into an action potential ORNs project their axons into the olfactory bulb of the brain 12 Odorant Receptors and the Organization of the Olfactory System 13 http://www.nobelprize.org/nobel_prizes/medicine/laureates/2004/press.html  Olfactory Receptor Signal Transduction Each odorant receptor detects one particular smell which activates an olfactory-specific heterotrimeric G-protein a-subunit, called Gaolf Gaolf stimulates the activity of an adenylyl cyclase isozyme, to generate cyclic AMP cAMP dependent ion channels open to allow influx of Na+ ions into the olfactory neuron – creating an action potential Other olfactory receptors are coupled to the phosphatidyl inositol pathway by heterotrimeric G- proteins, which is believed to stimulate IP3- or IP4- operated Ca2+ channels in the plasma membrane 14 Olfactory Receptor Signal Transduction Nature Reviews Neuroscience 11, 188-200 (March 2010) 15 Combinatorial Coding of Olfactory Information Figure 3. Combinatorial coding of olfactory information. Graphic representation of the olfactory receptor combinatorial code. In this hypothetical example, the responses of five odorant receptors to seven odorants (a–g) are shown, with the magnitudes of responses proportional to the sizes of the circles. Reflecting functional studies on individual odorant receptors, some receptors are more narrowly tuned than others, and individual odorants can activate different subsets (and numbers) of receptors. The pattern of receptor activation elicited by a particular compound is thought to represent that compound's chemical identity. DeMaria S , and Ngai J J Cell Biol 2010;191:443-452 16 Taste 17 18