CBS Membrane transport KEATS 23_24 PDF

Dr Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Department Biochemistry Membrane Transport  Teaching Objectives Describe the distinguishing features of small-molecule transfer ac...

Dr Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Department Biochemistry Membrane Transport  Teaching Objectives Describe the distinguishing features of small-molecule transfer across membranes by passive diffusion, facilitated transport and active transport Describe the structure and principle of action of the Na+/K+-ATPase membrane pump Describe the structure and principle of action of the Na+/glucose transporter protein family Describe the principle of action of the facilitated glucose transporter protein family Be aware of pathologies/treatments that involve membrane transport  Selective permeability Membranes are selective permeability barriers block passage of almost all hydrophilic molecules (into cells and organelles) Small uncharged or hydrophobic molecules can freely cross the membrane by simple diffusion along their concentration gradients Charged polar molecules require specialist proteins (pumps, transporters, pores) to allow them to across the membrane  Molecules crossing membranes Lipid Bilayer Hydrophobic O2, N2, CO2, benzene, molecules short chain fatty acids Small uncharged polar H2O, urea, molecules glycerol Large uncharged glucose, sucrose polar molecules Ions H+, Na+, Mg2+, HCO3- K+, Ca2+, Cl- Charged polar molecules amino acids, ATP Permeability of lipid bilayer is higher for molecules that are uncharged, non-polar, and small  Mechanisms of transport Simple passive transport / Diffusion Facilitated diffusion Gated ion channels (primary) Active transport Secondary active transport  Passive transport Solutes move down a concentration gradient crossing membrane At Equilibrium [inside cell] = [outside cell] Rate of diffusion depends on Partition Coefficient of solute Solutes that are more hydrophobic have a higher Partition Coefficient and equilibrate more quickly Example : H2O membrane OUT IN [So] [Si]  Facilitated diffusion / Carrier-mediated diffusion Solutes move down a concentration gradient crossing membrane At Equilibrium [inside cell] = [outside cell] Requires membrane protein (ion channel) Examples – Cl-/HCO3- channel in erythrocytes – aquaporin : water channel – GLUT glucose transporters  Cl-/HCO3- channel in erythrocytes Aqueous pore crossing membrane Biochemistry and Molecular Biology Fig7.14  Kinetics of transport : passive transport and facilitated diffusion J (Rate of ~Jmax uptake) Facilitated diffusion 1 /2Jmax Simple diffusion Km External concentration, [S]o  Transporter affinity Transporter affinity for solute is given by the Km The lower the Km the higher the affinity J (Rate of ~Jmax uptake) A B C 1 /2Jmax Affinity: A > B > C Km Km Km External concentration, [S]o  Facilitate diffusion of glucose Family of related glucose transporters (GLUT) Location Function GLUT1 Ubiquitous; Low Km ~ 1.8mM (high affinity). Mediates Abundant in constitutive glucose uptake in many tissues. erythrocytes, low in skeletal muscle GLUT2 Liver, pancreatic ß- High Km ~ 20mM (low affinity) and large Jmax cells (high capacity). Transports glucose into hepatocytes and pancreatic ß-cells when [glucose]blood is high to regulate blood glucose levels. GLUT3 Neurones Low Km (high affinity) GLUT4 Muscle, Adipocytes Km ~ 5mM : similar to Fed state blood glucose concentration. Regulated by insulin.  Insulin stimulated uptake of glucose Insulin stimulates uptake of glucose in muscle and adipose Insulin increases the amount of the GLUT4 in plasma membrane GLUT4 present on membrane bound vesicles in cytoplasm Insulin triggers the movement of vesicles to plasma membrane Vesicles merge with the plasma membrane and increase level on cell surface Increase in glucose transporters increases the uptake of glucose into the cell Recruitment of GLUT4 to membrane  Gated ion channels Ion channels that allow facilitated diffusion selective for different ions (K+ , Na + , Ca 2+) Open or close in response to stimulus Ligand-gated e.g. acetylcholine and acetylcholine-gated Na+/K+ channel (acetylcholine receptor) on postsynaptic membranes Voltage-gated e.g. Na+ and K+ channels in axons involved in nerve transduction Biochemistry and Molecular Biology Fig7.15 in axons  Active Transport Solutes move against a concentration gradient Requires membrane protein Requires energy – hydrolysis of ATP Primary active transport if ATP hydrolysis directly causes the movement of solute (uniport) Example – Na+/K+ pump (Na+/K+ ATPase) in plasma membrane membrane OUT IN  [Na+] and [K+] gradient across membrane Ion gradient of [Na+] and [K+] across plasma membrane [internal]cell : [Na+] ~12mM [K+] ~140mM [external]blood plasma : [Na+] ~145mM [K+] ~4mM Facilitates nerve transmission Part of co-transport system to drive solute movement Na+/K+ pump : function Biochemistry and Molecular Biology Fig7.11  Na+/K+ pump Na+/K+ pump consists of tetramer (α2β2) Na+ enter the open cytoplasmic access Phosphorylation from ATP at cytoplasmic site causes conformational change Conformational change closes cytoplasmic access and opens external access Conformational change means that the pump binds K+ and releases Na+ outside cell Hydrolysis of phosphate group closes external access, opens cytoplasmic access and releases K+ into cell  Co-transport systems Pre-established gradient is used to drive transport of solute across membrane against a gradient ATP hydrolysis is used to establish the primary gradient Symport : transport of two solutes in the same direction Antiport : transport of two solutes in opposite direction Uniport Symport Antiport  Na+ - glucose cotransporter (SGLUT) Glucose absorption from intestine against gradient SGLUT is a symport Na+ gradient established by the Na+/K+ pump and ATP hydrolysis is used to drive the uptake of glucose into cells Secondary active transport Similar system operates for uptake of amino acids Biochemistry and Molecular Biology Fig7.12  Role of glucose transporters in gut LUMEN OF THE GUT Apical Membrane (SGLUT) Tight Junction GLUCOSE Na+ K+ ATP ADP Basal Membrane GLUT2 Na+ BLOODSTREAM  Na+ / Ca2+ cotransporter Ca2+ export from muscle cells against gradient Na+ / Ca2+ cotransporter (sodium-calcium exchanger) is an antiport Na+ gradient established by the Na+/K+ pump and ATP hydrolysis is used to drive the export of Ca2+ from cells Secondary active transport Biochemistry and Molecular Biology Fig7.13  Clinical considerations : digitoxin Cardiac glycosides: digitoxin and digoxin, originally from fox-gloves, inhibit Na+/K+ pump by blocking the dephosphorylation step [Na+] inside heart muscle increases and the Na+ gradient is lost Export of Ca2+ via the antiport does not happen [Ca2+ ] inside heart muscle increases and contraction increases  Clinical considerations : ouabain Cardiac glycosides: ouabain, plant extract used in poison arrows, inhibit Na+/K+ pump by blocking binding of K+ [Na+] inside heart muscle increases and the Na+ gradient is lost Export of Ca2+ via the antiport does not happen [Ca2+ ] inside heart muscle increases and contraction increases https://en.wikipedia.org/wiki/Ouabain#/media/ File:Acokanthera_schimperi_-_K%C3%B6hler %E2%80%93s_Medizinal-Pflanzen-150.jpg  Clinical considerations : CFTR Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) CFTR is a chloride ion channel in cells responsible for producing mucus, sweat, saliva and tears Movement of chloride ions in turn regulates movement of water Patients have reduced chloride transport that results in the production of thick mucus This leads to blocked lungs and infections  CFTR Transmembrane protein Chloride ions move by facilitated diffusion down concentration gradient – out of cell ATP-gated ion channel ABC (ATP-binding cassette) transporter family Cytoplasmic regulatory domain is phosphorylated by cyclic AMP dependent protein kinase (PKA) PKA activates and opens channel  Clinical considerations : cholera treatment Vibrio cholerae causes electrolyte and fluid secretion Cholera toxin stimulates increase in cAMP level that activates CFTR and secretion of chloride ions Na+ and water follow into lumen of via osmosis and the paracellular route Oral rehydration therapy includes high glucose concentration (~110 mM) which drives Na+ (and consequently Cl- and H2O) uptake into cells via SGLUT  Summary Selective permeability of membranes Simple passive transport / Diffusion Facilitated diffusion – GLUT Gated ion channels (primary) Active transport – Na/K pump Secondary active transport – symport and antiport – SGLUT