Passive vs. active transport

1. Passive vs. active transport • Passive transport is simply transport down an electrochemical gradient until equilibrium is reached • Active transport results in accumulation of solute beyond equilibrium, the unfavorable thermodynamics is driven by ATP hydrolysis

2. Passive vs. facilitated diffusion

3. Four types of transport ATPases • F-type – you are familiar with • P-type • V-type • Multi-drug transporter (ABC Transporter)

4. P-type ATPase • Cation transporter that is reversibly phosphorylated as part of the transport cycle • Vanadate sensitive • Na+, K+, Ca++ • Bacteria use to detoxify heavy metals • Widely distributed

5. A P-type ATPase maintains potassium and sodium gradient

6. This transport mechanism maintains a membrane potential

7. Ion gradients provide energy for secondary active transport • Ion gradients formed by transport of cations can be driving force for cotransport of other solutes

8. For example,

9. A composite look at transport

10. V-type ATPase and Multi-drug transporter • V-type • Works as a proton pump • Has key role in acidification of cellular compartments (including endosome) • Multi-drug transporter • Export numerous compounds in ATP-dependent manner

11. Ion channels are distinct from ion transporters • Ion channels provide a faster rate of transport • Ion channels cannot be saturated • Channels are gated, meaning they are open or closed in response to allosteric effectors

12. Channel structure provides insight into specificity and rate

13. Features of K+ channel • Negatively charged amino acids act as sink for cations • Pathway narrows (filter) to accommodate interactions specific for potassium • Appears to be a paradigm for ion channels (calcium, etc.)

14. Mechanosensitive channels • MscS

15. Structural facets of MscS • ~120 Angstroms in length (membrane portion ~50 and extramembrane ~70) • The extramembrane region of MscS (cytoplasmic) surrounds a large water-filled chamber of ~40 Å diameter • Three transmembrane helices, kink at residue 113 marks membrane boundary (what residue?) • Surprisingly, the electrostatic potential of transmembrane region is positive (arginines and lysines), prefer to transport anions to cations • Specific arginine residues appear poised for sensing changes in membrane potential • However, at 3.9 angstrom resolution one cannot tell where all the side chains point and H-bonds cannot be discerned

16. How does it work?

17. Apparent amino acid requirements of MscS • Current model suggests movement of TM1 and TM2 in respect to TM3 between open and closed conformation • Mechanosensitive channels – little known about sequence requirements, small amino acids (Gly or Ala) at specific positions are considered important to accommodate changes in helix packing • Voltage gating – some well characterized examples (Kv family of channels) with six TM helices. Positive charges spaced out along specific TM (S4) which is loosely packed against remainder of TM

18. Voltage gated sodium channels Change in membrane potential results in conformational change

19. Lastly, Ligand-gated channels • Acetylcholine receptor binds acetylcholine causing a conformational change opening the ion channel. • There are also intracellular ligand-gated channels

20. Ligand gated ion channels • Nicotinic acetylcholine receptor transports sodium, calcium and potassium ions through conformational changes

21. Membrane proteins such as channels, receptors have significant metabolic roles • Hormones and metabolites offer signaling or communication mechanisms within the cell

22. Qualities of Signal Transduction

23. Molecular cascades

24. Feedback inhibition

25. Integrated networks

26. Six types of signal transducers

27. Other two receptor types • Receptors with no intrinsic enzyme activity (can interact with enzymes though such as tyrosine kinases) to affect gene expression • Adhesion receptor, binds molecules in the extracellular matrix

28. Looking at receptor-ligand interactions • Experimentally must account for non-specific binding (ie. to the membrane, tube, etc.)

29. Scatchard analysis (like Lineweaver Burke)

30. Case study: Receptor Enzymes • Most commonly – • tyrosine kinases

31. Insulin receptor and a regulatory cascade

32. Important facets of this process • Phosphorylation alters protein structure/function • SH2 domain observed in many proteins, a conserved domain that mediates protein-protein interactions • G-protein activates kinases • Kinases act in a cascade to modulate transcriptional regulators (kinome)

33. IRS-1 can interact with other cellular components for network integration

34. G-proteins and signal transduction • b-adrenergic Signal pathway

35. How? • Epinephrine binds the serpentine receptor, causing GTP to replace GDP on the G-protein (this particular G-protein is distinct from Ras family) • G becomes active with GTP bound, and activates adenyl cyclase, which converts ATP to cAMP • Timing mechanism turns off G protein

37. cAMP activated protein kinases • Protein kinase A

39. Result of epinephrine cascade

40. Densensitization by phosphorylation

41. Second messenger cAMP

42. Other second messengers • Diacylglycerol • Inositol triphosphate • Calcium