Cell Membranes and Membrane Transport PDF 2022

Faculty of Life Sciences and Medicine Professor Christer Hogstrand School of Cancer and Pharmaceutical Sciences Cell Biology and Neuroscience Membranes and membrane transport Internal membranes 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 2 The Fluid Mos...

Faculty of Life Sciences and Medicine Professor Christer Hogstrand School of Cancer and Pharmaceutical Sciences Cell Biology and Neuroscience Membranes and membrane transport Internal membranes 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 2 The Fluid Mosaic Model of membrane structure ‘Proteins floating in a sea of lipids’ Carbohydrates Phospholipids 4-5nm Proteins 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 3 Biological membranes are selective permeability barriers Biological membranes act as selective permeability barriers that block the passage of almost all water soluble molecules (in and out of cells and organelles) Small uncharged or hydrophobic (lipid soluble) molecules can freely traverse the bilayer by simple diffusion down their concentration gradients Charged polar molecules require specialist proteins (pumps, transporters, pores) to carry them across the membrane 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Permeation of different solutes across a lipid bilayer Hydrophobic molecules O2, N2, benzene, short chain fatty acids Small uncharged polar molecules H2O, CO2, urea, glycerol Large uncharged polar molecules glucose, sucrose Ions H+, Na+, Mg2+, HCO3K+, Ca2+, Cl- Charged polar molecules amino acids, ATP Permeability of lipid bilayer is higher for molecules that are uncharged, non-polar, and small Lipid Bilayer 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 5 Concentrations of ions and charges are different on the inside of cells from the outside Ion [IN] mM [OUT] mM Na+ 10 140 K+ 140 4 Cl- 4 140 0.0001 2.5 Ca2+ Proteins Membrane Potential ψ 12/10/2022 Professor Christer Hogstrand --- + Topic title: Cell Membranes and Membrane Transport 6 Transport mechanisms Needs carrier protein? Simple Facilitated Primary diffusion diffusion Active Transport Secondary Active Transport No Yes Yes Yes With Against Against No No Yes (ATP hydrolysis) Yes, Electrochemical gradient H2 O Glucose (GLUT) With or against concentration With gradient? Energy Required? Examples 12/10/2022 Professor Christer Hogstrand Na+/K+ATPase Topic title: Cell Membranes and Membrane Transport Na+/Glucose transporter (intestine) 7 Simple / Passive diffusion Solute moves from one side of the membrane to the other along its concentration gradient OUT [So]o IN [So]i [So]o > [So]i OUT [So]o IN [So]i [So]o = [So]i Rate of diffusion depends on the octanol/water PARTITION COEFFICIENT (Kow) of the solute 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 8 Octanol-Water partitioning coefficient Kow, the equilibrium constant for partitioning of a molecule between oil (octanol) and water The higher the value for Kow the more lipid soluble it is e.g. diethyl urea has a Kow = 0.01, urea has a Kow = 0.0002 Diethyl urea is 50 times more hydrophobic and travels through a lipid bilayer 50 times faster than urea 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Transmembrane movement of ions and hydrophilic molecules is mediated by proteins e.g. ion channels, water channels (aquaporins) 12/10/2022 Professor Christer Hogstrand Figure 11-3b Molecular Biology of the Cell (© Garland Science 2008) Topic title: Cell Membranes and Membrane Transport PROFESSORS PETER AGRE & RODERICK MACKINNON WON NOBEL PRIZE IN 2003 FOR “DISCOVERIES CONCERNING WATER AND ION CHANNELS” http://nobelprize.org 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport The K+ channel, KCSA, from the bacterium Streptomyces lividans Ion Selectivity Filter H2O Amino acid side chains in ion filter 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport http://nobelprize.org Ion channels are usually gated Figure 11-21 Molecular Biology of the Cell (© Garland Science 2008) 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Facilitated diffusion can involve a conformational change in a selective carrier protein CONC GRADIENT 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Transport of one solute / coupled transport UNIPORT SYMPORT Coupled transport ANTIPORT 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport The kinetics of transport distinguish between simple diffusion and carrier mediated transport J (Rate of uptake) Jmax Carrier mediated 1/ J 2 max Simple Km 12/10/2022 Professor Christer Hogstrand External concentration [S]o Topic title: Cell Membranes and Membrane Transport Transporter affinity for substrate is given by the Km J (Rate of uptake) 1/ J 2 max Jmax A B C Affinity: A > B > C Km Km Km External concentration, [S]o 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport The lower the Km of a transporter for its substrate, the higher the affinity Facilitated glucose transporters Facilitated glucose transport is mediated by a family of 12 distinct transporters (GLUT1-12), which show sequence homology but tissue specific distribution. Gene/Protein Approximate Km Location Features SLC2A1 / GLUT1 Ubiquitous; Highly expressed in blood-brain-barrier, erythrocytes 1 – 2 mM Basal glucose uptake SLC2A2 / GLUT2 Liver, kidney, intestinal epithelium, pancreatic ß-cells 15 – 20 mM Glucose sensing and transport when blood [glucose] is high SLC2A3 / GLUT3 Neurons 1 – 2 mM Basal glucose uptake SLC2A4 / GLUT4 Muscle, adipocytes 5 mM Regulated by insulin Gorovits and Charron (2003) BIOCHEM MOL BIOL EDU 31:3, 163-172 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Overall structure and working model of the human glucose transporter GLUT1 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Deng et al. (2014) Nature 510: 121-125 19 Regulation of GLUT4 by insulin INSULIN Binds to membrane receptor Signals (phosphorylation cascade) to intracellular pool of GLUT4 12/10/2022 Professor Christer Hogstrand GLUT4 Translocation of GLUT4 to membrane When Insulin levels fall GLUT4 recycled to pool Topic title: Cell Membranes and Membrane Transport Active transport OUT IN Movement of solute against the concentration gradient; This required ENERGY PRIMARY – HYDROLYSIS OF ATP SECONDARY – ELECTROCHEMICAL GRADIENT 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Primary active transport Energy supplied by the hydrolysis of ATP ATP ADP + Pi This drives energetically unfavourable biochemical processes Na+/K+-ATPase in plasma membrane H+-ATPase in lysosomal membrane Ca2+-ATPase in plasma membrane; endoplasmic/sarcoplasmic reticulum 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Functioning of the Na+/K+-ATPase – Step 1 HIGH [Na+] LOW [K+] OUT P IN Na+ High Affinity Na+ Binding Site ATP LOW [Na+] HIGH [K+] ADP 1. Na+ BINDS TO INTRACELLULAR SITE 2. THIS TRIGGERS AN AUTOPHOSPHORYLATION OF THE PUMP 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 23 Functioning of the Na+/K+-ATPase – Step 2 OUT Na+ K+ HIGH [Na+] LOW [K+] P IN LOW [Na+] HIGH [K+] 1. PHOSPHORYLATION CAUSES A CONFORMATIONAL CHANGE TO RELEASE Na+ TO THE EXTERIOR AND TO EXPOSE A K+ BINDING SITE 2. BINDING OF K+ TRIGGERS DEPHOSPHORYLATION OF THE PUMP 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 24 Functioning of the Na+/K+-ATPase – Step 3 HIGH [Na+] LOW [K+] OUT LOW [Na+] HIGH [K+] IN K+ PUMP RETURNS TO ORIGINAL CONFORMATION AND K+ IS DISCHARGED INTO THE INTERIOR OF THE CELL WHOLE PROCESS ~10 ms 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Functioning of the Na+/K+-ATPase – overall cycle HIGH [Na+] Na+ Binding Site K+ and ouabain binding site (and plant glycosides e.g. digitalis) 3Na+ ATP ADP + Pi 2K+ HIGH [K+] An “electrogenic” process 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport The Na+/K+-ATPase is a drug target for treatment of congestive heart failure In the heart muscle, the Na+/Ca2+ antiporter (NCX) contributes to removal Ca2+ from the cytosol allowing cardiac relaxation to occur. 1. Oubain inhibits the Na+/K+-ATPase by preventing K+ binding, decreasing the rate of Na+ extrusion from cardiac muscle cells. 2. This increases the intracellular [Na+], which reduces the activity of NCX, resulting in slower Ca2+ efflux and therefore prolonged high cytosolic [Ca2+] to maintain cardiac muscle contraction. Na+ 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport Ca2+ Ca2+ 27 Purple foxglove, first used by William Withering in 1785 Digitalis purpurea William Withering FRS (1741 –1799) Source of digitoxin and digoxin – cardiotonic drugs, increase contraction 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 28 Secondary active transport OUT HIGH [Na+] LOW [GLUCOSE] IN LOW [Na+] HIGH [GLUCOSE] Na+ GLUCOSE 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 29 SGLT1 and SGLT2 are sodium-glucose symporters UTILISATION OF AN ELECTROCHEMICAL GRADIENT e.g. Na+/Glucose Transporters (SGLT1 and SGLT2) IMPORTANT IN: 1. SGLT1: Intestinal epithelial cells for the absorption of dietary glucose Medical Physiology A Cellular and Molecular Approach, Updated 2nd Ed. 2. SGLT2: Epithelial cells in proximal tubules of the kidney for reabsorption of glucose from the primary urine 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 30 Glucose uptake by intestinal epithelial cells LUMEN OF THE GUT Apical Membrane (NB also GLUT5 here) SGLT1 Na+ GLUCOSE ATP Basal Membrane GLUT2 Tight Junction K+ ADP Na+/K+-ATPase Na+ BLOODSTREAM 12/10/2022 Professor Christer Hogstrand Topic title: Cell Membranes and Membrane Transport 31 Cholera toxin, produced by Vibrio cholerae causes huge electrolyte and fluid losses in the intestine 1. Cholera toxin (CT) binds to the GM1 ganglioside receptor on the apical membrane of intestinal cells 2. CT is internalised by endocytosis and transported through the Golgi to the ER 4. 5. 12/10/2022 In the ER subunits of the toxin are split and the A1 subunit escapes to the cytosol. H2O ClCFTR A + B A ADP-ribose This activates the CFTR Cl- channel leading to large Cl- secretion Na+ follows the electrical gradient 7. H2O follows the osmotic gradient Professor Christer Hogstrand GT P B A1 binds to and overactivates the heterotrimeric GTPase Gsa, leading to an activation of Adenylyl Cyclase and an increase in cAMP levels 6. Tight junction cAMP A Adenylyl Cyclase Gas 3. Na+ g ß A B A ER B Topic title: Cell Membranes and Membrane Transport Lencer, Wayne. 2001. Am J Physiol. Gastrointest Liver Physiol. 280: G781-G786. 32 Role of the Na+/Glucose Cotransporter in Electrolyte Replacement Therapy in Cholera SGLUT Replacement therapy includes a high concentration of glucose (~110 mM), which drives Na+ (and consequently Cl- and H2O) back into the intestine through the Na+/glucose symporter, SGLT1 Cl- 12/10/2022 Topic title: Cell Membranes and Membrane Transport Professor Christer Hogstrand Na+ Glucose H2O 33 Thanks for your attention Whenever possible, use KEATS for questions [email protected]