Membranes II

1. Membranes II Andy HowardIntroductory Biochemistry6 October 2009 Biochemistry: Membranes II

2. Membranes work hard • Transport of various types requires active participation of various proteins and sometimes involves energy input. • Interactions between signaling molecules and receptors occurs at the membrane and allow an external signaling molecule to influence internal behavior. Biochemistry: Membranes II

3. Membrane transport Review Transporting charges Pores & Channels Passive Transport Active Transport Moving large molecules Signal transduction General Principles G proteins Adenylyl cyclase Inositol-phospholipid signaling pathway Receptor tyr kinases What we’ll discuss Biochemistry: Membranes II

4. Cartoons of transport types • From accessexcellence.org Biochemistry: Membranes II

5. 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. 9.4 through 9.6. Biochemistry: Membranes II

6. 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 Biochemistry: Membranes II

7. 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 Biochemistry: Membranes II

8. 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. Biochemistry: Membranes II

9. 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 Biochemistry: Membranes II

10. 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 Biochemistry: Membranes II

11. Pores and channels • 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+) Rod MacKinnon Biochemistry: Membranes II

12. 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 Biochemistry: Membranes II

13. 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. Biochemistry: Membranes II

14. 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. Biochemistry: Membranes II

15. Velocity versus [S]out Vmax = 0.5 mM s-1 Ktr = 0.1 mM Biochemistry: Membranes II

16. 1/v0 versus 1/[S]out Biochemistry: Membranes II

17. 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 Biochemistry: Membranes II

18. Secondary active transport • Active transport of one solute 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 Biochemistry: Membranes II

19. 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 Biochemistry: Membranes II

20. 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 Biochemistry: Membranes II

21. 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 Biochemistry: Membranes II

22. 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 Biochemistry: Membranes II

23. Example: LDL-cholesterol Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences Biochemistry: Membranes II

24. 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 Biochemistry: Membranes II

25. Transducing signals • Plasma membranes contain receptors that allow the cell to respond to chemical stimuli that 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 Biochemistry: Membranes II

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

27. 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 Biochemistry: Membranes II

28. Internal results of signals • Intracellular: heterotrimeric G-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 Biochemistry: Membranes II

29. 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! Biochemistry: Membranes II

30. G proteins • 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 Biochemistry: Membranes II

31. GTP Inactive GDP G protein cycle a Active b a g GTP b • Ternary complex disrupted by binding of receptor complex • Ga-GTP interacts with effector enzyme • GTP slowly hydrolyzed away • Then Ga-GDP reassociates with b,g • See fig. 9.39 for details H2O g Pi a GDP Inactive Biochemistry: Membranes II

32. Adenylyl cyclase Cyclic AMP • cAMP and cGMP: second messengers • Adenylyl cyclase converts ATP to cAMP • Integral membrane enzyme; active site faces cytosol • cAMP diffuses from membrane surface through cytosol, activates protein kinase A • Protein Kinase A (PKA) phosphorylates ser,thr in target enzymes;action is reversed by specific phosphatases Biochemistry: Membranes II

33. Modulators of cAMP • Caffeine, theophylline inhibit cAMP phosphodiesterase, prolonging cAMP’s stimulatory effects on protein kinase A • Hormones that bind to stimulatory receptors activate adenylyl cyclase, raising cAMP levels • Hormones that bind to inhibitory receptors inhibit adenylyl cyclase activity via receptor interaction with the transducer Gi. Biochemistry: Membranes II

34. Inositol-Phospholipid Signaling Pathway • 2 Second messengers derived from phosphatidylinositol 4,5-bisphosphate (PIP2) • Ligand binds to specific receptor; signal transduced through G protein called Gq • Active form activates phosphoinositide-specific phospholipase C bound to cytoplasmic face of plasma membrane PIP2 Biochemistry: Membranes II

35. PIP2 chemistry • Phospholipase C hydrolyzes PIP2 to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol • Both of these products are second messengers that transmit the signal into the cell Biochemistry: Membranes II

36. IP3 and calcium • IP3 diffuses through cytosol and binds to a calcium channel in the membrane of the endoplasmic reticulum • The calcium channel opens, releasing Ca2+ from lumen of ER into cytosol • Ca2+ is a short-lived 2nd messenger too: it activates Ca2+-dependent protein kinases that catalyze phosphorylation of certain proteins Biochemistry: Membranes II

37. Diacylglycerol and protein kinase C • Diacylglycerol stays @ plasma membrane • Protein kinase C (which exists in equilibrium between soluble & peripheral-membrane form) moves to inner face of membrane; it binds transiently and is activated by diacylglycerol and Ca2+ • Protein kinase C catalyzes phosphorylation of several proteins Biochemistry: Membranes II

38. Control of inositol-phospholipid pathway • After GTP hydrolysis, Gq is inactive so I no longer stimulates Plase C • Activities of 2nd messengers are transient • IP3 rapidly hydrolyzed to other things • Diacylglycerol is phosphorylated to form phosphatidate Biochemistry: Membranes II

39. Spingolipids give rise to 2nd messengers • Some signals activate hydrolases that convert sphingomyelin to: • sphingosine • sphingosine-1-P, and • ceramide • Each of these modulates a second messenger Biochemistry: Membranes II

40. Fates of the three sphingolipid products • Sphingosine inhibits Protein Kinase C • Ceramides activate a protein kinase and a protein phosphatase • Sphingosine-1-P can activate Phospholipase D, which catalyzes hydrolysis of phosphatidylcholine;products are 2nd messengers Biochemistry: Membranes II

41. ligands exterior Receptor tyrosine kinases Tyr kinase monomers interior • Most growth factors function via a pathway that involves these enzymes • In absence of ligand, 2 nearby tyr kinase molecules are separated • Upon substrate binding they come together, form a dimer Biochemistry: Membranes II

42. Autophosphorylation of the dimer P P • Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation • Once it’s phosphorylated, it’s activated and can phosphorylate various cytosolic proteins, starting a cascade of events Biochemistry: Membranes II

43. Insulin receptor • Insulin binds to an a2b2 tetramer;binding brings b subunits together • Each tyr kinase (b) subunit phosphorylates the other one • The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation Sketch courtesy ofDavidson College, NC Biochemistry: Membranes II