《生物化學(xué)第十一章》ppt課件

1、Ediden, M. (2003) Nat. Rev. Mol. Cell Biol. 4: 414 - 41815. 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

2、 gradient2 TransportersCarriesChannels 23UniportersSecondaryactivetransportersPrimaryactivetransporters1What is the difference between carrier and channel ?3The Transporter Classification System1.A. a Helix type channels voltage-gated K+ channel aquaporins acetylcholine receptor/channel1.B. b Barrel

3、 porins general bacterial porin (GBP) family1.C. Pore-forming toxins2.A. Porters: uniporters, symporters, and antiporters 2.B. Nonribosomally synthesized porters: valinomycin3.A. Diphosphate bond hydrolysis-driven transporters (use PPi, not ATP) : ATP-binding cassette (ABC) superfamily P-type ATPase

4、 superfamily F-, V- and A-type ATPase superfamily4Concentration GradientsThe 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

5、 G = RTln C2/C15Charge GradientsIn 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 6Passive D

6、iffusion 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 kineticsPassiveFacilitatedA(outside)A(inside)V0 = kA0kA(outside) + MP (membrane protein)A(MP)A(inside) + MPkV0

7、= Vmax A0/ (Kt + A0 )7A 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 8Aquaporins form hydrophilic transmembrane channels forthe passage of water9 Water mol

8、ecules 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 protons10Aquaporins form hyd

9、rophilic transmembrane channels for the passage of watereach monomer: 6 transmembrane helices4 monomer: forming the channel, through which water can diffuse NPA-containing short helix11Structure of an aquaporin, AQP-1tetramerpore6 helices/monomer1 monomnerAsn-Pro-Ala (NPA)water hydrophilic atomsCons

10、triction created byside chains of Phe58, His182,Cys191, Arg197.Phe58Arg197 and His182 residues and electric dipoles formed by NPA loops provide positive charges in positions that repel any protons. 12Osmosis - a special case of diffusion, also passive. occurs when membranes are permeable to water bu

11、t not to dissolved ions and small polar organic solutes. The movement of solvent from regions of to high solute concentration. may manifest as volume change (until solute conc. is equalized) pressure changeH2Osemi-permeablemembrane13If the concentration of water in the medium surrounding a cell is g

12、reater than that of the cytosol, the medium is said to be hypotonic (低滲， hypertonic，高滲). 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 hyp

13、otonic surroundings by their strong cell walls. These allow the buildup of turgor (膨壓) within the cell. When the turgor pressure equals the osmotic pressure, osmosis ceases.14glucose transporter: mediates passive transportGluT1: 12 hydrophobic segments (membrane-spanning helix)15A helical wheel diag

14、ram shows the distribution of polar and nonpolar residues on the surface of a helical segmentSide-by-side association of 5-6 amphipathic helicesproduce a transmembrane channel16Kinetics of glucose transport into erythrocytesGluT1: high rates ; saturability; specificity 17Model of glucose transport i

15、nto erythrocytes by GluT1T1: glucose binding site exposed on the outer surface of PM T2: glucose binding site exposed on the inner surface of PMk1k2k3Sout + T SoutT Sin T Sin + Tk-1k-2k-3V0 = Vmax S0ut/ (Kt + Sout )18Defective glucose and water transport in two forms of diabetes1. GluT4 (myocytes &

16、adipocytes)Insulin interacts with insulin receptor movement of intracellular vesicles (GluT4) to PM glucose uptake 2. Aquaporin (AQP2), regulated by ADH (antidiuretic hormone) defect in AQP2 water re-absorption by kidney 19Regulation by insulin of glucose transport into a myocyte20chloride-bicarbona

17、te exchanger (anion exchange protein, facilitated diffusion)- Increases the permeability of the erythrocyte membrane to HCO3- by a factor of more than a million;- an example of cotransport system;- Allows the entry and exit of HCO3- without changes in the transmembrane electrical potential.21Against

18、 chemical and electrical gradient : Gt = RT ln (C2/C1) + Z FR: gas constant, 8.315 J/mol KT: absolute temperatureZ: charge of ion: transmembrane electrical potential F: Faraday constant (96,48o J/V mol)22Four general types of transport ATPase ATP dependent active transporterP-type ATPaseV-type ATPas

19、e F-type ATPase Multidrug transporter Different in structure, mechanism and location 23P-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

20、cytosol2425Na+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%

21、 in neurons).P-type:26Na+K+ ATPase-discovered by Jens Skou in 1957;-Integral protein with two subunits27Mechanism of Na+ and K+transport by Na+K+ ATPaseFormation of phosphoenzyme: ATP + EnzI ADP + P-EnzIIHydrolysis: P-EnzII + H2O EnzI + PiThe net reaction : ATP + H2O ADP + Pi28Vanadate (phosphate an

22、alog): inhibitor of P-type ATPasePalytoxin (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.29 potent and specificinhibitor of the Na+K+ ATPaseOuabain and digitoxigeninare the active ingredients of digitalis, which has b

23、een used as medicine to treat heart failure.binds preferentiallyto the form of enzymethat is open to the extracelluarside30The Nobel Prize in chemistry in 1997for his discovery of Na+K+ ATPase Jens SkouDenmark 1918-31Sarcoplasmic reticulum32Mechanism of SR Ca2+ ATPase Action33Reversibility of F-type

24、 ATPase: play a central role in energy-conserving reactions in bacteria, mitochondria, and chloroplast34ABC (ATP-binding cassette) transporters use ATP to drive the active transport of a wide variety of substratesABC transporters: ATP-dependent transporters;Pump amino acids, peptides, proteins, meta

25、l ions, various compounds including drugs (MDR1: multidrug transporter) out of cells against a concentration gradient.NBD: nucleotide- binding domainLipid A flippaseVitB12 importer35CFTR: cystic fibrosis transmembrane conductance regulator: ion channel specific for Cl-(defect: cystic fibrosis, delet

26、ion of Phe508)ATP binding site(deletion)The structure is relatedto MDR1 transporter.36In CF patients: high concentration of NaCl in surface fluid,which is less effective in killing bacteriaMucus lining the surface of the lungs traps bacteria37Secondary Active Transport(powered by primary active tran

27、sport)38What is relationship between primary active transport andsecondary active transport ?39Three general classes of transport systems40Secondary Active Transport Processes41Lactose uptake in E.coliPrimary transportSecondary transportCN- (cyanate): inhibit the primary transport42Structure of the

28、lactose transporter (lactose permease) of E. coli (Ron Kaback and So Iwata in 2003)The mechanism for transmembrane passage of the substrate involvesa rocking motion between the two domains, driven by substrate binding and proton movement (protonation of side chains of Glu325 and Arg302).Rocking Bana

29、na Model lactoseGlu325 , Arg30243Glucose is cotransported with Na+ across the apical PM into the epithelial cellPrimary transport: Na+K+ ATPase ;Secondary transport(symport): 44The energy required comes from two sources: Chemical potential ; Transmembrane potential (electrical potential) Energy prod

30、uced in primary transportG = 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 kJG = -22.5 kJ = RT ln glucose in/glucose outglucose in/glucose out = 9000“Symport” : pump glucose inward until its concentration withinthe epithelial cell is 9000 times than in the i

31、ntestine.45Valinomycin : 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.46 Ion channels can be highly selective for particular ions. Unsaturable an

32、d 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

33、regulated. Ion channels are divided in two classes: Ligand-gated channels Voltage-gated channels47The structure of a K+ channel shows the basisfor its ion specificitytetramer of 4 identical subunits8 transmembrane helicesK+48 Selective Filter of the K+ Channel(K+ interact with the carbonyl groups of

34、 the high conserved sequence of the selective filter, located at the 3 diameter pore of the channel.)Ionic radiusK+: 1.4 Na+: 0.95 Backbone carbonyl oxygens form cage that fits K+ precisely, replacing waters of hydration sphere. ExtracellularspaceCytosolK+ with hydrating water molecules water-filled

35、 vestibule allows hydration of K+ Helix dipole stabilizes K+Alternating K+ sites (blue or green) occupied49There are four K+ binding sites along the selectivity filter,each composed of an oxygen “cage” that provides ligandsfor the K+ ions.Carbonyl oxygenMovement of the two K+ ions is concerted: firs

36、t they occupy position1 and 3, then hop to position 2 and 4.The energetic difference betweenthese two configurations is very small. The combined effect of K+ binding andrepulsion between K+ ions ensures that an ion keeps moving in a maximal flow with high specificity. 123450Question: How is the high

37、 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 providingcompensating interactions with the carbonyl oxygen atoms lining the selective filter. However, these oxygen atoms ar

38、e positioned such that they do not interact favorably with Na+ (too small). 51The 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

39、 structural and mechanistic studies of ion channels” Howard Hughes Medical Institute, The Rockefeller University, New York, USA 52Two scientists , Montal & Mueller, contributed an experimental system that demonstrated for the first time that purified membrane proteins could be reconstituted to full

40、functionality and recapitulate their biological function. Their contribution was first for the nicotinic acetylcholine receptor, the voltage-gated sodium channel, the photosynthetic reaction centers, cytochrome oxidase, and later on the bacterial toxin channels, Bcl-2 family of proteins, and the HIV

41、 Vpu proteins. Ligand-gated channel: acetylcholine receptor53 Essential in the passage of an electrical signal from a motor neuron to a muscle fiber at neuromuscular junction; Acetylcholine binds its receptor conformational change of the receptor inward movement of Na+, Ca 2+ and K+; Structure of ac

42、etylcholine receptor: five subunits (a2b), each subunit consists of four transmembrane helices, M1, M2 (amphipathic), M3 and M454a-subunit55Leu side chainSmall and polar residuesAcetylcholine binds its receptor causes conformational change of the receptor56Inhibitor of acetylcholine receptor57Voltag

43、e-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

44、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 channel58ball-and-chain mechanism 59H

45、elix 4Voltage-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 60Ball-and-Chain MechanismInactivation gate (ball) is tethered to th

46、e 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.61Inhibitors of voltage-gated Na+ channels62Electrical measurements o

47、f ion channel functionDetection:104 ions/millisecond- developed by Ervin Neher and Bert Sakmann in 197663Porins are transmembrane channels for small molecules64An ion transporter (porin) in E. Coli.: Fhu A bring ferrichrome from extracellular medium across outer membrane to the periplasmic space; co

48、mposed of 22-b domain and “cork” domain (N-terminus)open or close6566 Summary1. Composition and architecture of membranesl definition of biological membranesl composition of membranesl membrane proteins: peripheral and integral proteinsl characters of membrane proteinsl the lipid and membrane proteins are inserted into the bilayer with specific sidedness.2. Membrane dynamics l Affect of lipids to membrane fluidityl Flip-flop diffusion of lipids67l Lipids and proteins can diffuse laterally within the plane of the membrane, but this mobility is