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Membranes, transport, and signaling

This lecture explores the various ways molecules move across membranes and how signals are transmitted across them. Topics include membrane proteins, membrane transport, passive and active transport, and signal transduction.

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Membranes, transport, and signaling

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  1. Membranes, transport, and signaling Andy HowardIntroductory BiochemistryWednesday 8 October 2014 Membranes, Transport, Signaling

  2. Membranes: transport and signaling • We’ll complete our discussion of the ways that things move across membranes, and then talk about the way that signals pass across membranes. Membranes, Transport, Signaling

  3. Membrane proteins Membrane transport Neutral molecules Charges Pores & Channels Passive Transport Active Transport Moving large molecules Signal transduction General Principles G proteins Adenylyl cyclase Inositol-phospholipid signaling pathway Sphingolipid messages Receptor tyrosine kinases What we’ll discuss Membranes, Transport, Signaling

  4. Membrane Proteins (G&G §9.2) • Many proteins associate with membranes • But they do it in several ways • Integral membrane proteins:considerable portion of protein is embedded in membrane • Peripheral membrane proteins:polar attachments to integral membrane proteins or polar groups of lipids • Lipid-anchored proteins:protein is covalently attached via a lipid anchor Membranes, Transport, Signaling

  5. Integral(Transmembrane) Proteins Drawings courtesy U.Texas • Span bilayer completely • May have 1 membrane-spanning segment or several • Often isolated with detergents • 7-transmembrane helical proteinsare very typical (e.g. bacteriorhodopsin) • Beta-barrels with pore down the center: porins Membranes, Transport, Signaling

  6. Peripheral Membrane proteins • Also called extrinsic proteins • Associate with 1 face of membrane • Associated via H-bonds, salt bridges to polar components of bilayer • Easier to disrupt membrane interaction:salt treatment or pH Chloroflexus auracyanin 15.4 kDa PDB 1QHQ1.55Å Membranes, Transport, Signaling

  7. Lipid-anchored membrane proteins • Protein-lipid covalent bond • Often involves amide or ester bond to phospholipid • Others: cys—S—isoprenoid (prenyl) chain • Glycosyl phosphatidylinositol with glycans Membranes, Transport, Signaling

  8. N- Myristoylation & S-palmitoylation Membranes, Transport, Signaling

  9. Membrane Transport • What goes through and what doesn’t? • Nonpolar gases (CO2, O2) diffuse • Hydrophobic molecules and small uncharged molecules mostly pass freely • Charged molecules blocked Membranes, Transport, Signaling

  10. Transmembrane Traffic:Types of Transport (Table 9.3) Type Protein Saturable Movement Energy Carrier w/substr. Rel.to conc. Input? Diffusion No No Down No Facilitated Yes No Down No diffusion* Passive Yes Yes Down No transport Active^ Yes Yes Up Yes* accomplished primarily through pores and channels^ two kinds: primary and secondary Membranes, Transport, Signaling

  11. Cartoons of transport types • From accessexcellence.org Membranes, Transport, Signaling

  12. Thermodynamics ofpassive and active transport • If you think of the transport as a chemical reaction Ain Aout or Aout Ain • It makes sense that the free energy equation would look like this: • Gtransport = RTln([Ain]/[Aout]) • More complex with charges;see eqns. 9.1 through 9.2 Membranes, Transport, Signaling

  13. Example • Suppose [Aout] = 145 mM, [Ain] = 10 mM,T = body temp = 310K • DGtransport = RT ln[Ain]/[Aout]= 8.325 J mol-1K-1 * 310 K * ln(10/145)= -6.9 kJ mol-1 • So the energies involved are moderate compared to ATP hydrolysis Membranes, Transport, Signaling

  14. Charged species • Charged species give rise to a factor that looks at charge difference as well as chemical potential (~concentration) difference • Most cells export cations so the inside of the cell is usually negatively charged relative to the outside Membranes, Transport, Signaling

  15. Quantitative treatment of charge differences • Membrane potential (in volts  J/coul):DY = Yin - Yout(there’s an extra D in eqn. 9.4) • Gibbs free energy associated with difference in electrical potential isDGe = zFDYwhere z is the charge being transported and F is Faraday’s constant, 96485 JV-1mol-1 • Faraday’s constant is a fancy name for 1. Membranes, Transport, Signaling

  16. Faraday’s constant • Relating energy per moleto energy per coulomb: • Energy per mole of charges,e.g. 1 J mol-1, is1 J / (6.022*1023 charges) • Energy per coulomb, e.g, 1 V = 1 J coul-1, is1 J / (6.241*1018 charges) • 1 V / (J mol-1) =(1/(6.241*1018)) / (1/(6.022*1023) = 96485 • So F = 96485 J V-1mol-1 Membranes, Transport, Signaling

  17. Total free energy change • When charges move, we typically have both a chemical potential difference and an electrical potential difference so • DGtransport = RTln([Ain]/[Aout]) + zFDY • Sometimes these two effects are opposite in sign, but not always Membranes, Transport, Signaling

  18. Pores and channels Rod MacKinnon • Transmembrane proteins with centralpassage for small molecules,possibly charged, to pass through • Bacterial: pore. Usually only weakly selective • Eukaryote: channel. Highly selective. • Usually the DGtransport is negative so they don’t require external energy sources • Gated channels: • Passage can be switched on • Highly selective, e.g. v(K+) >> v(Na+) Membranes, Transport, Signaling

  19. Gated potassium channels • Eukaryotic potassium channels are gated, i.e. they exist in open or closed forms • When open, they allow K+ but not Na+ to pass through based on ionic radius (1.33Å vs. 0.95Å) • Some are voltage gated; others are ligand gated Membranes, Transport, Signaling

  20. Protein-facilitated passive transport • All involve negative DGtransport • Uniport: one solute across • Symport: two solutes, same direction • Antiport: two solutes, opposite directions • Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow • These proteins can be inhibited, reversibly or irreversibly Diagram courtesy Saint-Boniface U. Membranes, Transport, Signaling

  21. Kinetics of passive transport • Michaelis-Menten saturation kinetics:v0 = Vmax[S]out/(Ktr + [S]out) • We’ll derive that relationship in the enzymatic case in a later chapter • Vmax is velocity achieved with fully saturated transporter • Ktr is analogous to Michaelis constant:it’s the [S]out value for which half-maximal velocity is achieved. Membranes, Transport, Signaling

  22. Velocity versus [S]out Vmax = 0.5 mM s-1 Ktr = 0.1 mM Membranes, Transport, Signaling

  23. 1/v0 versus 1/[S]out Membranes, Transport, Signaling

  24. Primary active transport • Energy source is usually ATP or light • Energy source directly contributes to overcoming concentration gradient • Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production • P-glycoprotein: ATP-driven active transport of many nasties out of the cell Membranes, Transport, Signaling

  25. Secondary active transport • Active transport of onesolute is coupled to passive transport of another • Net energetics is (just barely) favorable • Generally involves antiport • Bacterial lactose influx driven by proton efflux • Sodium gradient often used in animals Membranes, Transport, Signaling

  26. Complex case: Na+/K+ pump • Typically [Kin] = 140mM, [Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM. • ATP-driven transporter:3 Na+ out for 2 K+ inper molecule of ATP hydrolyzed • 3Na out: 3*6.9 kJmol-1,2K in: 2*8.6 kJmol-1= 37.9 kJ mol-1 needed, ~ one ATP Diagram courtesy Steve Cook Membranes, Transport, Signaling

  27. What’s this used for? • Sodium gets pumped back in in symport with glucose, driving uphill glucose transport • That’s a separate passive transport protein called GluT1 Diagram courtesy Steve Cook Membranes, Transport, Signaling

  28. How do we transport big molecules? • Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis • Special types of lipid vesicles created for transport Membranes, Transport, Signaling

  29. Receptor-mediated endocytosis • Bind macromolecule to specific receptor in plasma membrane • Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside) • Vesicle fuses with endosome and a lysozome • Inside the lysozyome, the foreign material and the receptor get degraded • … or ligand or receptor or both get recycled Membranes, Transport, Signaling

  30. Example: LDL-cholesterol Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences Membranes, Transport, Signaling

  31. Exocytosis Diagram courtesy LinkPublishing.com • Materials to be secreted are enclosed in vesicles by the Golgi apparatus • Vesicles fuse with plasma membrane • Contents released into extracellular space Membranes, Transport, Signaling

  32. Transducing signals • Plasma membranes containreceptors that allow the cellto respond to chemical stimulithat can’t cross the membrane • Bacteria can detect chemicals:if something useful comes along,a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source Image courtesy Nobelprize.orgfrom 1994 P&M award to Gilman & Rodbell Membranes, Transport, Signaling

  33. Multicellular signaling • Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals Diagram courtesy Science Creative Quarterly, U. British Columbia Membranes, Transport, Signaling

  34. Extracellular Signals • Internal behavior ofcells modulated by external influences • Extracellular signals are called first messengers • 7-helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors Image courtesy CSU Channel Islands Membranes, Transport, Signaling

  35. Internal results of signals • Intracellular: heterotrimericG-proteins are the transducers: they receive signal from receptor, hydrolyze GTP, and emit small molecules called second messengers • Second messengers diffuse to target organelle or portion of cytoplasm • Many signals, many receptors, relatively few second messengers • Often there is amplification involved Membranes, Transport, Signaling

  36. Roles of these systems • Response to sensory stimuli • Response to hormones • Response to growth factors • Response to some neurotransmitters • Metabolite transport • Immune response • This stuff gets complicated, because the kinds of signals are so varied! Membranes, Transport, Signaling

  37. G proteins (G&G § 32.4) • Transducers of external signals into the inside of the cell • These are GTPases (GTP  GDP + Pi) • GTP-bound protein transduces signalsGDP-bound protein doesn’t • Heterotrimeric proteins; association of b and g subunits with a subunit is disrupted by complexation with hormone-receptor complex, allowing departure of GDP & binding of GTP Membranes, Transport, Signaling

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