LMP 200H – Lecture 4 Calcium PDF

LMP 200H – Lecture 4 Calcium Jan. 2025 Calcium has unique roles: Resting cytosolic Ca2+ ca. 100 nM Signals in two ways: Uptake of extracellular ion Release from ER/SR stores Both harmful and essential - regulates cell death and survival, causes injury and pe...

LMP 200H – Lecture 4 Calcium Jan. 2025 Calcium has unique roles: Resting cytosolic Ca2+ ca. 100 nM Signals in two ways: Uptake of extracellular ion Release from ER/SR stores Both harmful and essential - regulates cell death and survival, causes injury and performs numerous essential functions. Injury Mechanism – Increased Intracellular Ca Ca2+ and apoptosis i) Ca2+ activates key catabolic enzymes Proteases – calpain, nuclear scaffold protease (NS) Endonucleases – NUC18, DNase I Transglutaminases – stabilize structures and/or target for degradation Actin-binding proteins, e.g., gelsolin Ca2+ and apoptosis ii) Ca2+ depletion destabilizes ER – release of NS, DNase I Mitochondrion – drop in  Some functions of intracellullar Ca2+: Signal transduction Excitation-contraction coupling Cytoskeletal remodeling Apoptosis Calcium homeostasis: Resting cytosolic Ca2+ ca. 100 nM Signals in two ways: Uptake of extracellular ion Release from ER/SR stores Some players: Uptake – Voltage- & Ligand-gated channels, capacitative entry & CRAC Release – IP3R and Ryanodine Receptors (RyR) Expulsion – plasma membrane pumps & exchangers Resequestration – P-type ATPase, SERCA  Phospholipase A1 H2CO C R1 Phospholipase A2 O R2 C OCH Phospholipase D Arachidonic acid O O R2 often unsaturated. H2CO P R3 (e.g., IP2) Peroxide more susceptible to PL A2. O- Phospholipase C IP3 PIP2  IP3 receptors PLC –> IP –> activates Ca release 3 2+ Family of peptides ~300 kDa Two transmembrane regions anchored to ER Aggregate to tetramers to form Ca channels 2+ Stimulated by Ca (amplification), inhibited by caffeine 2+ Some isoforms of IP receptors interact with CRAC 3  Ryanodine receptors (RyR) C-terminal transmembrane regions homologous to IP R3 ~565 kDa, extra regulatory domains Also form tetramer RyR1 (skeletal muscle), RyR2 (cardiac muscle) Calmodulin binding domain, triggered by Ca influx 2+ E-C coupling through calmodulin/MLCK Stimulated by caffeine RyR1 cytoplasmic tail associates with DHP receptor DHP as voltage sensor, also ligand gated (cADP ribose?) Calcium-charged Calmodulin Calmodulin – a Ca2+ sensor - 148 a.a. - 4 Ca2+-binding sites, in E-F hand structures (E & F helices of parvalbu Roles in: – E-C coupling (MLC Kinase) – Metabolism (phosphorylase kinase – glucose, calcitonin – lipids) – Neurochemistry (CaMK II) A calmodulin "switch" Fill and Copello, 2002 Malignant hyperthermia (high fever) Susceptibility in pigs: Stress-induced muscle contraction, leads to hyperthermia and death Porcine RyR1 has R615C mutation, fails to close properly, becomes hyperexcitable  MH in humans Inhalational (volatile) anaesthetics (halothane, isoflurane,…). Most cases associated with RyR1 mutations (>25). Incidence 1/5,000 - 1/50,000 with anaesthetic. Testing: Muscle biopsy with caffeine-halothane test. Tx: Dantrolene (muscle relaxant), cooling. Death: 5 % with Tx, 80 % without. Mechanisms in malignant hyperthermia Depletion of Ca2+ stores: Ca2+ mobilizing agents IP3 Ionophores (e.g., ionomycin) ER pump inhibitors (e.g., thapsigargin) Replenishment of Ca2+ stores: Capacitative entry Conformational coupling/IP3 with CRAC (analogy to RyR1/DHPR) Slow entry, CRAC Privileged pathways Slow entry linked to mitogenic respones, increased in SV40-transformed cells, defect leads to immunodeficiency.  1.1 x 104 ions/s Calsequestrin, Calreticulin 1.5 x 106 ions/s  Ca2+ oscillations Excitable cells, secretory cells (e.g., pancreatic  cell) ~memb –> ~VOC, but also in Ca2+-free medium Avoid sustained increases, e.g. –> apoptosis More information: integrate both frequency and amplitude Possible mechanisms: Oscillating IP3 via G protein/receptor/PLC, PKC inactivates G protein or receptor, … Two-pool calcium-induced calcium release (CICR), with an IP3-insensitive capacitative pool that fills first. IP3-independent, e.g., RyR-sensitive stores, ~memb, … Maintenance of Ca2+ gradients Core Ca2+ signaling network Panel (A) Cytoplasmic [Ca2+] kept at ~100 nM by extrusion via plasma membrane Ca2+ ATPase (PMCA) and smooth endoplasmic reticular Ca2+ ATPase (SERCA). Na/Ca exchanger is a major secondary regulator of [Ca2+] and is electrogenic. Intracellular Ca2+ hyperpolarizes cells by activating K+ (and Cl−) channels. This decreases CaV channel activity but increases the driving force across active Ca2+-permeant channels. Panel (B) In excitatory Ca2+ signaling, plasma membrane ion channels are triggered by changes in voltage, or extra- or intracellular ligand binding. ~1 million Ca2+ ions/s/channel flow down the 2x104-fold [Ca2+]i gradient (ECa ~ +150 mV). Initial increases in [Ca2+] trigger more release, primarily from ER via RyR. GPCR- or RTK-mediated activation of PLC cleaves PIP2 into IP3 and DAG. GPCRs catalyze the exchange of GDP for GTP on Gα subunits, releasing active Gα and Gβγ subunits that in turn activate PLCβ. RTKs dimerize upon ligand binding, autophosphorylate, and interact with other signaling proteins to activate PLCγ. Voltage-operated Ca2+ Channels Elliott Nicholson: https://www.youtube.com/watch?v=erYKVbAgZCo https://www.youtube.com/watch?v=-61b2EY-V9Y Use of Ca2+ channel blockers in hypertension: Vasorelaxation, decreased calmodulin activation and MLC phosphorylation More active in arterial than venous SMC, decreased afterload Reversal of endothelin-mediated vasoconstriction Effective in virtually all patients if cardiac complications permit Use of Ca2+ channel blockers in MI: Decrease cardiac O2 demand decrease heart rate (sinus node) decrease afterload (vasodilation) decrease contractility (-ve inotropic effect) decrease response to ET-1 (limits use of Ca-ATPase pump) Vasorelaxant effect, decreases post-MI hypertension Vasodilatation of coronary arteries Decreased excitability, antiarrhythmic effect Use of Ca2+ channel blockers in CHF: Meta-analysis of clinical trials discouraging -ve inotropic effect with borderline ejection fraction Depressed troponin C - Ca2+ interaction inhibits contraction DHPs may reset baroreceptor, increase beating rate in vivo, though decrease it in cultured myocytes Chronic increase in plasma norepinephrine