《生物化学第十一章》PPT课件.ppt

Ediden, M. (2003) Nat. Rev. Mol. Cell Biol. 4: 414 - 418 5. Membrane transport: Proteins can facilitate or drive the transport of specific molecules across a membrane barrier - Passive transport Diffusion Facilitated transport - Active transport Energy-coupled system ATP powered transport Concentration gradient Classification of transportersClassification of transporters Transporters Carries Channels 23 UniportersSecondary active transporters Primary active transporters 1 What is the difference between carrier and channel ? The Transporter Classification System 1.A. Helix type channels voltage-gated K+ channel aquaporins acetylcholine receptor/channel 1.B. Barrel porins general bacterial porin (GBP) family 1.C. Pore-forming toxins 2.A. Porters: uniporters, symporters, and antiporters 2.B. Nonribosomally synthesized porters: valinomycin 3.A. Diphosphate bond hydrolysis-driven transporters (use PPi, not ATP) : ATP-binding cassette (ABC) superfamily P-type ATPase superfamily F-, V- and A-type ATPase superfamily Concentration Gradients The conc. gradient is the difference in conc. between each compartment (C2-C1) The chemical potential difference (G) for a molecule diffusing from one side with C1 to the other with C2 defined by the ratio of the concentrations G = RTln C2/C1 Charge Gradients In addition to conc. gradient, a difference in charge can lead to electrostatic attraction that may drive diffusion Z = charge on molecule being diffused F = Faraday constant (96,485 JV-1 mole-1) = voltage difference (electric potential) G = RTln C2/C1 + ZF Passive Diffusion Passive diffusion is a first order kinetic process and rate depends directly on concentration. Facilitated diffusion is assisted by proteins and displays Michaelis-Menten reaction kinetics Passive Facilitated A(outside)A(inside) V0 = kA0 k A(outside) + MP (membrane protein) A(MP)A(inside) + MP k V0 = Vmax A0/ (Kt + A0 ) A transporter protein reduces the G for transmembrane diffusion of the solute: - by forming non-covalent interactions with the hydrated solute - providing a hydrophilic transmembrane passageway Aquaporins form hydrophilic transmembrane channels for the passage of water Water molecules flow through an APQ-1 channel at the rate of 109 s-1. The low activation energy for passage of water through aquaporin channel (G 15kJ/mol) is required. In the direction dictated by osmotic gradient; Selectivity: create constriction, select water molecules but not protons Aquaporins form hydrophilic transmembrane channels for the passage of water each monomer: 6 transmembrane helices 4 monomer: forming the channel, through which water can diffuse NPA-containing short helix Structure of an aquaporin, AQP-1 tetramer pore 6 helices/monomer 1 monomner Asn-Pro-Ala (NPA) water hydrophilic atoms Constriction created by side chains of Phe58, His182, Cys191, Arg197. Phe58 Arg197 and His182 residues and electric dipoles formed by NPA loops provide positive charges in positions that repel any protons. Osmosis - a special case of diffusion, also passive. occurs when membranes are permeable to water but not to dissolved ions and small polar organic solutes. The movement of solvent from regions of lowlow solutesolute concentration concentration to high solute concentration. may manifest as volume change (until solute conc. is equalized) pressure change H2O semi-permeable membrane solutesolute HH 2 2 OO If the concentration of water in the medium surrounding a cell is greater than that of the cytosol, the medium is said to be hypotonic (低渗， hypertonic，高渗). Water enters the cell by osmosis. A red blood cell placed in a hypotonic solution (e.g., pure water) bursts immediately (“hemolysis“) from the influx of water. Plant cells and bacterial cells avoid bursting in hypotonic surroundings by their strong cell walls. These allow the buildup of turgor (膨压) within the cell. When the turgor pressure equals the osmotic pressure, osmosis ceases. glucose transporter: mediates passive transport GluT1: 12 hydrophobic segments (membrane-spanning helix) A helical wheel diagram shows the distribution of polar and nonpolar residues on the surface of a helical segment Side-by-side association of 5-6 amphipathic helices produce a transmembrane channel Kinetics of glucose transport into erythrocytes GluT1: high rates ; saturability; specificity Model of glucose transport into erythrocytes by GluT1 T1: glucose binding site exposed on the outer surface of PM T2: glucose binding site exposed on the inner surface of PM k1k2k3 Sout + T SoutT Sin T Sin + T k-1k-2k-3 V0 = Vmax S0ut/ (Kt + Sout ) Defective glucose and water transport in two forms of diabetes 1. GluT4 (myocytes - an example of cotransport system; - Allows the entry and exit of HCO3- without changes in the transmembrane electrical potential. Active transport:Active transport: solute movement against solute movement against concentration concentration and and electrical potentialelectrical potential (electrochemical gradient)(electrochemical gradient) Against chemical and electrical gradient : Gt = RT ln (C2/C1) + Z F R: gas constant, 8.315 J/mol K T: absolute temperature Z: charge of ion : transmembrane electrical potential F: Faraday constant (96,48o J/V mol) Four general types of transport ATPase ATP dependent active transporter P-type ATPase V-type ATPase F-type ATPase Multidrug transporter Different in structure, mechanism and location P-type: ATP-driven cation transporter (phosphorylated by ATP); V-type: proton pumps, V for vesicles or vacuolar; F-type (ATP synthase): proton pumps, catalyze ATP formation; Multidrug transporter: removes drugs from cytosol Na+K+ ATPase K+ is needed by cells to activate many processes; But, Na+ inhibits these processes; Gradient of Na+ and K+ (or other ions) drive secondary active transport processes for amino acids, sugars, nucleotides, etc. 20% to 40% of a cells ATP used to maintain these ion gradient (70% in neurons). P-type: Na+K+ ATPase -discovered by Jens Skou in 1957; -Integral protein with two subunits Mechanism of Na+ and K+ transport by Na+K+ ATPase Formation of phosphoenzyme: ATP + EnzI ADP + P-EnzII Hydrolysis: P-EnzII + H2O EnzI + Pi The net reaction : ATP + H2O ADP + Pi high affinityhigh affinity for Nafor Na + + high affinityhigh affinity for Kfor K + + Vanadate (phosphate analog): inhibitor of P-type ATPase Palytoxin (水螅毒素)：binds to the enzyme and lock it into a position in which the ion-binding sites are permanently accessible from both sides. OuabainOuabain (“arrow poison”): (“arrow poison”): potent and specific inhibitor of the Na+K+ ATPase Ouabain and digitoxigenin are the active ingredients of digitalis, which has been used as medicine to treat heart failure. OuabainOuabain DigitoxigeninDigitoxigenin binds preferentially to the form of enzyme that is open to the extracelluar side The Nobel Prize in chemistry in 1997 “for his discovery of Na+K+ ATPase “ Jens Skou Denmark 1918- P-type CaP-type Ca2+ 2+ pump pump ( (Sarcoplasmic reticulum): ): maintain a low maintain a low concentration of Caconcentration of Ca2+ 2+ in in the the cytosolcytosol (0.1 (0.1 mMmM), ), compare with Cacompare with Ca2+ 2+ in SR in SR is 1.5 is 1.5 mMmM Mechanism of SR Ca2+ ATPase Action Reversibility of F-type ATPase: play a central role in energy-conserving reactions in bacteria, mitochondria, and chloroplast ABC (ATP-binding cassette) transporters use ATP to drive the active transport of a wide variety of substrates ABC transporters: ATP-dependent transporters; Pump amino acids, peptides, proteins, metal ions, various compounds including drugs (MDR1: multidrug transporter) out of cells against a concentration gradient. NBD: nucleotide- binding domain Lipid A flippaseVitB12 importer CFTR: cystic fibrosis transmembrane conductance regulator: ion channel specific for Cl- (defect: cystic fibrosis, deletion of Phe508) ATP binding site (deletion) The structure is related to MDR1 transporter. In CF patients: high concentration of NaCl in surface fluid, which is less effective in killing bacteria Mucus lining the surface of the lungs traps bacteria Secondary Active Transport (powered by primary active transport) Active transportActive transport What is relationship between primary active transport and secondary active transport ? Three general classes of transport systems The classification does not tell us whether The classification does not tell us whether the transport is active or passivethe transport is active or passive Secondary Active Transport Processes Lactose uptake in E.coli Primary transport Secondary transport CN- (cyanate): inhibit the primary transport Structure of the lactose transporter (lactose permease) of E. coli (Ron Kaback and So Iwata in 2003) The mechanism for transmembrane passage of the substrate involves a rocking motion between the two domains, driven by substrate binding and proton movement (protonation of side chains of Glu325 and Arg302). Rocking Banana Model lactose Glu325 , Arg302 Glucose is cotransported with Na+ across the apical PM into the epithelial cell Primary transport: Na+K+ ATPase ; Secondary transport (symport): 2Na2Na + + outout + glucose + glucose out out 2Na2Na + + inin + glucose + glucose in in SymportSymportUniportUniport The energy required comes from two sources: Chemical potential ; Transmembrane potential (electrical potential) Energy produced in primary transport G = n RT ln Na+in/Na+out + Z F G = 2 x 8.315 x 310 ln (12/145) + (-2x 96.480 x 50) = - 22.5 kJ G = -22.5 kJ = RT ln glucose in/glucose out glucose in/glucose out = 9000 “Symport” : pump glucose inward until its concentration within the epithelial cell is 9000 times than in the intestine. Valinomycin : a peptide ionophore that binds K+. Both valinomycin and monensin (Na+ ionophore) are antibiotics; they kill microbiol cells by disrupting secondary transport processes and energy-conserving reactions. Ion-Selective ChannelsIon-Selective Channels Ion channels can be highly selective for particular ions. Unsaturable and have very high flux rates; Ion channels exist in open and closed state. These channels undergo transition from the closed state, incapable of supporting ion transport, to the open state, through which ions can flow (very rapid, milliseconds). Transitions between the open and the closed states are regulated. Ion channels are divided in two classes: Ligand-gated channels Voltage-gated channels The structure of a K+ channel shows the basis for its ion specificity tetramer of 4 identical subunits 8 transmembrane helices K+ Selective Filter of the K+ Channel (K+ interact with the carbonyl groups of the high conserved sequence of the selective filter, located at the 3 diameter pore of the channel.) Ionic radius K+: 1.4 Na+: 0.95 Backbone carbonyl oxygens form cage that fits K+ precisely, replacing waters of hydration sphere. Extracellular space Cytosol K+ with hydrating water molecules water-filled vestibule allows hydration of K+ Helix dipole stabilizes K+ Alternating K+ sites (blue or green) occupied There are four K+ binding sites along the selectivity filter, each composed of an oxygen “cage” that provides ligands for the K+ ions. Carbonyl oxygen Movement of the two K+ ions is concerted: first they occupy position 1 and 3, then hop to position 2 and 4. The energetic difference between these two configurations is very small. The combined effect of K+ binding and repulsion between K+ ions ensures that an ion keeps moving in a maximal flow with high specificity. 1 2 3 4 Question: How is the high degree of selectivity achieved ? The 3 diameter filter rejects ions having a radius larger than or smaller than 1.5 ; Reasons: The channels pays the cost of dehydrating K+ by providing compensating interactions with the carbonyl oxygen atoms lining the selective filter. However, these oxygen atoms are positioned such that they do not interact favorably with Na+ (too small). The Nobel Prize in Chemistry 2003 “ for discoveries concerning channels in cell membranes” Peter Agre “for the discovery of water channels” School of Medicine Johns Hopkins University Baltimore, USA Roderick MacKinnon “for structural and mechanistic studies of ion channels” Howard Hughes Medical Institute, The Rockefeller University, New York, USA Two scientists , Montal Acetylcholine binds its receptor conformational change of the receptor inward movement of Na+, Ca 2+ and K+; Structure of acetylcholine receptor: five subunits (2), each subunit consists of four transmembrane helices, M1, M2 (amphipathic), M3 and M4 -subunit Leu side chain Small and polar residues Acetylcholine binds its receptor causes conformational change of the receptor Inhibitor of acetylcholine receptor Voltage-gated channel: neuronal Na+ channel The channels exist in the plasma membranes of neurons and of myocytes of heart and skeletal muscle; The channels sense electrical gradient across the membrane and respond by opening or closing; Very selective for Na+ and have a very high flux rate; Structure: a single, large polypeptide organized into four domains, each containing 6 transmembrane helices; Roles of different helices: helix 4: voltage sensor; helix 5: selective filter; helix 6: activation and inactivation gate ball-and-chain mechanism: inactivation of the channel ball-and-chain mechanism Helix 4 Voltage-Sensing Mechanism - Helix 4 response to change in transmembrane potential; - Membrane polarized: Helix 4 is pulled inward; - Membrane depolarized: Helix 4 relaxes by moving outward; - Communication (interact) with helix 6 Ball-and-Chain Mechanism Inactivation gate (ball) is tethered to the channel by a short segment of polypeptide (the chain). The “ball” domain is free to move when the channel is closed, when it is open, a site on the inner face of the channel is available for the ball to bind, blocking the channel. Inhibitors of voltage-gated Na+ channels Electrical measurements of ion channel function Detection: 104 ions/millisecond - developed by Ervin Neher and Bert Sakmann in 1976 Porins are transmembrane channels for small molecules An ion transporter (porin) in E. Coli.: Fhu A bring ferrichrome from extracellular medium across outer membrane to the periplasmic space; composed of 22- domain and “cork” domain (N-terminus) open or close Summary of Transport TypesSummary of Transport Types Summary 1. Composition and architecture of membranes l definition of biological membranes l composition of membranes l membrane proteins: peripheral and integral proteins l characters of membrane proteins l the lipid and membrane proteins are inserted into the bilayer with specific sidedness. 2. Membrane dynamics l Affect of lipids to membrane fluidity l Flip-flop diffusion of lipids l Lipids and proteins can diffuse laterally within the plane of the membrane, but thi