Transporters and Membrane Motors Nov 15, 2007

Transcription

1 BtuB OM vitamin B12 transporter F O F 1 ATP synthase Human multiple drug resistance transporter P-glycoprotein Transporters and Membrane Motors Nov 15, 2007

2 Transport and membrane motors Concentrations of solutes and transmembrane gradients Functions of channels and transporters Bioenergetics of concentration and ionic gradients How are ionic gradients used: the Proton Motive Force Mitochondria, Bacteria, Chloroplasts Oxidative (photo) phosphorylation e - transport chain ATP synthesis Transport defined Channels vs transporters Passive (or facilitated) vs active transport Molecular features: porins ion channels facilitated transporters 1 transporters 2 transporters

3 [Ca 2+ ] 1.2 mm free, 2.5 mm total 1.2 mm 10-7 M free [Mg 2+ ] 0.6 mm free, 0.9 mm total 0.55 mm 1 mm free, 18 mm total

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5 DIFFUSION A B A B B A Net flux

6 Osmotic behavior of cells in solutions of permeating and non-permeating solutes Cells behave as ideal osmometers in solutions of impermeant solutes 154 mm NaCl (an impermeant solute) is isotonic (no cell volume change) > 154 mm NaCl is hypertonic (cells shrink) < 154 mm NaCl is hypotonic (cells swell) tonic refers to the steady state or equilibrium situation

7 Ionic Effects A B + - K + =1M K + =0.1M + - Concentration Electrical

8 Introduce an electric potential of -60 mv (A relative to B). Which way will the ion flow? A K + =1 M mv B K + =0.1 M Concentration Electrical

9 Electrochemical potential»» energy term A B μ = μ o + RT lnc + zf E K + =1 M K + =0.1 M Energy/mole Elec. μ A = μ o + RT lnc A + zf E A - + μ B = μ o + RT lnc B + zf E B E A -E B = -60 mv Concentration Electrical C A μ A -μ B = Δμ = RT ln + zf (E A -E B ) C B Conc. Force Elec. force

10 Oxidative phosphorylation is carried out by a series of coupled transporters The enzymes of the mitochondrial inner membrane involved in oxidative phosphorylation. NADH-dehydrogenase (yellow), succinate dehydrogenase (pink), cytochrome bc1 (red), and cytochrome oxidase (green) form the electron transfer chain to O2. With the exception of SDH, these enzymes translocate protons across the membrane. The proton gradient is used by ATP synthase (purple) to make ATP. From Saraste, M. Science 283:

11 Chemiosmotic coupling PMF (proton motive force) = electrochemical gradient of protons = Δμ H+ = zfδψ + RT ln [H+ ] i [H + ]o electrical chemical Matrix space Membrane Potential, ΔΨ H Inner membrane Matrix space Chemical Potential, ΔpH H + H + H + H + H + H + H + H + H + H + H + H + Inner membrane

12 Δμ H+ is used to drive many chemiosmotic coupled systems

13 Structure of F and V-type ATPases F 1 F O ATP synthase from E. coli Vacuolar ATPase from yeast Component sectors Assembled active form From Nelson and Harvey, Physiol. Rev. 79: , 1999

14 Animation of ATP synthesis Wolfgang Junge

15 Protein-mediated transport Rapid relative to diffusion Saturation kinetics Except channels Specificity Chemical Stereochemical Competitive inhibition Non-competitive inhibition J [S] facilitated diffusion

16 Facilitated transport vs channel Properties of channels Permeation-Channels do not saturate Very fast ions per second Selective-some have strong preference for certain ion species Sometimes gated-open or close in response to certain stimuli: voltage, ligands, mechanical, etc. J channel facilitated diffusion [S]

17 Facilitated vs Active transport Facilitated transporters are not linked to energy Active transporters are linked to energy, either directly (1 ) or indirectly (2 ) If not energy linked, then downhill transport only Example: glucose transport L-glucose not transported Galactose, arabinose compete Phloretin - non competitive inhibitor Insulin stimulates Phloretin

18 Porins - mitochondrial, chloroplast, and bacterial outer membranes only. Generally non-selective but size limited

19 Water channels aquaporins glycerol permeability facilitator

20 Ion channels

21 Pores vs channels

22 Closed and opened conformations of the pore From MacKinnon (2003) FEBS Lett. 555, 62-65

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25 The voltage-gated K+ channel KvAP. Helical elements S3b and S4 form a hydrophobic `voltage sensor paddle' with gating charge arginine residues. From MacKinnon (2003) FEBS Lett. 555, 62-65

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27 Major structural features of the known families of ion channels

28 Is Cl - in equilibrium? A B For Cl - to be in equilibrium: Cl - =1 M Cl - =0.1 M E A -E B = +100 mv E A -E B must = 60 mv -1 E Cl = +60 mv log 10 1 M 0.1 M So E A -E B is in the right direction but is larger than it needs to be. Thus electrical force > concentration force And Cl - is not in equilibrium Which direction will Cl - flow? From B A

29 Active transporters Uphill Vectorial Energetics Types of active transporters Structure and function of active transport F and V-type P-type ABC-type Secondary transporters

30 Primary (1º) transporters in animal cells Cu

31 Active transport for uphill movement of a substance Transports substance against an electrochemical gradient high μ x low μ x

32 Active transport requires energy Where does the energy come from? ATP directly - Primary transport From the electrochemical gradient From movement of another substance, i.e., Na + - Secondary transport Metabolic inhibitors will eventually inhibit transport as [ATP] is run down

33 Transport is vectorial Transported solute binds to a site on one side with a particular affinity The orientation of the site changes to other side Upon changing orientation, the affinity of the site becomes low and the solute is released This process takes energy

34 Energetics of primary transport Δμ x = μ xa - μ xb, if only the chemical concentration gradient is considered: Δμ x = -nrt ln [x] A [x] B if ATP is the energy source, get ~10-15 kcal/mol -RT ln [x] A [x] B = kcal/mol if 1 ion is transported per ATP consumed, can drive a gradient of : [x] A [x] = ~ B if 2 ions are transported per ATP consumed, can drive a gradient of: [x] A [x] A - (2) RT ln [x] = kcal/mol = ~ B [x] B

35 Structures of SERCA in different conformations Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues CHIKASHI TOYOSHIMA, HIROMI NOMURA* & TAKEO TSUDA Nature, 2004, 432,

36 Movie of transport cycle

37 Topology of P-glycoprotein, a multiple drug resistance ABC transporter From Ambudkar et al. Oncogene (2003) 22,

38 Structure of ABC transporters Structure of MsbA. Views of MsbA in the open (left) and closed (right) conformation looking into the chamber opening (side view) and from the extracellular side (top view). The TMD, ICD, and NBD are colored red, dark blue, and cyan, respectively. The loop connecting the TMD with the ICD observed in the closed conformation from V. cholerae (right) is highlighted in green and the loop connecting the and domain of the NBD is shown in orange. The approximate position of the membrane bilayer is indicated by black lines. From Chang, G. (2003) FEBS Lett. 555,

39 Secondary (2º) transporters in mammalian cells Note that these transporters are coupled to an electrochemical gradient driven by a 1 transporter, i.e. Na + These are examples of co-transporters, or symporters

40 2 transporters-exchangers or anti-porters

41 Structure of 2 transporters-lacy and GlpT From Abramson et al. 2003, Science 301, (LacY), and, Huang et al Science 301, (GlpT)