Cellular Processes

1. Cellular Processes Diffusion, channels and transporters

2. Cellular Membranes • Two main roles • Allow cells to isolate themselves from the environment, giving them control of intracellular conditions • Help cells organize intracellular pathways into discrete subcellular compartment, including organelles

3. Membrane Structure • Lipid bi-layer: phospholipids, primarily phosphoglycerides • Other lipids • Sphingolipids: alter electrical properties • Glycolipids: communication between cells • Cholesterol: increase fluidity while decreasing permeability Figure 3.20

4. Membrane Proteins • Can be more than half of the membrane mass • Two main types • Integral membrane proteins – tightly bound to the membrane, either embedded in the bilayer or spanning the entire membrane • Peripheral proteins – weaker association with the lipid bilayer We will discuss membrane proteins that allow for flow of ions or for transport of molecules

5. Membrane permeability Lipid bilayer

6. Membrane proteins we will discuss

7. Membrane Transport • Three main types • Passive diffusion • Facilitated diffusion • Active transport

8. Passive Diffusion • Lipid-soluble molecules (alcohol, CO2) • No specific transporters are needed • No energy is needed • Depends on concentration gradient • High  low • Steeper gradient results in higher rates • Gradients can be chemical, electrical or both depending on the nature of the molecule • e.g., Membrane potential – electrical gradient across a cell membrane

9. Facilitated Diffusion • Hydrophilic molecules • Protein transporter is needed - Uniporter • No energy is needed • Depends on concentration gradient • Examples: amino acids, nucleosides, sugars (glucose)

10. Facilitated Diffusion, Cont. • Three main types of proteins 1. Ion channels – form pores, channel has to be open • Open/close in response to a membrane potential b) Open via specific regulatory molecules c) Regulated through interactions with subcellular proteins

11. Facilitated Diffusion, Cont. • 2. Porins – like ion channels, but for larger molecules • Cool stuff: aquaporin allows water to cross the plasma membrane – 13 billion H2O molecules per second! But, as pointed out by T. Todd Jones that is only 0.000000000000018 ml of water. • 3. Permeases – function more like an enzyme. Binds the substrate and then undergoes a conformation change which causes the carrier to release the substrate to the other side. Ex. Glucose permeases

12. Facilitated Diffusion - Uniporter GLUT1 – mammalian glucose transporter • Uses concentration gradient of glucose to drive transport • Can work in reverse • Used by most mammals

13. Electrical Gradients • All transport processes affect chemical gradients • Some transport processes affect the electrical gradient • Electroneutral carriers: transport uncharged molecules or exchange an equal number of charged particles • Electrogenic carriers: transfer a charge, e.g., Na+/K+ ATPase  exchanges 3Na+ for 2K+

14. Membrane Potential • Difference in charge inside and outside the cell ↔ electrochemical gradient • Active transporters establish this gradient • Two main functions • Provide cell with energy for membrane transport • Allow for changes in membrane potential used by cells in cell-to-cell signaling Can be determined by Nernst equation and Goldman equation

15. Nernst equation • Used to calculate the electrical potential at equilibrium • Recall:ΔG = RTln([Xi]/[Xo]) + zFEm • Chemical component + electrical component • At equilibrium:zFEm = RTln([Xo]/[Xi]) • Equilibrium potential is: • Ex = (RT/zF) ln [Xo]/[Xi] • where R – gas constant, T = absolute temperature (Kelvin), • z = valence of ion, F – Faradays constant • Example: K+ out: 0.01 M; K+ in: 0.1 M; T = 22oC • So, EK+ = (1.9872*295)/(1*23062) ln (0.01/0.1) • = -58 mV at 22oC

16. Nernst equation Each ion has a different potential given the difference in concentration gradients. Must have pores or channels to create potential!

17. Nernst equation and ion concentrations Differences in Nernst potential reflect differences in chemical gradients! We will discuss the protein pumps that are necessary to maintain these gradients.

18. Active Transport • Protein transporter is needed • Energy is required • Molecules can move from low to high concentration

19. Active Transport, Cont. • Two main types: distinguished by the source of energy • Primary active transport – uses an exergonic reaction ie ATP • Secondary active transport – couples the movement of one molecule to the movement of a second molecule

20. Primary Active Transport • Hydrolysis of ATP provides energy • Three types • P-type: pump specific ions, e.g., Na+, K+, Ca2+ • F- and V-type: pump H+ • ABC type: carry large organic molecules, e.g., toxins

21. P-class pumps: Na+/K+ ATPase pump • pumps 2 K+ in and 3 Na+ out important for many cellular functions (osmotic balance of cells) uses ATP as energy source can be blocked with poisons like ouabain or digitalis the potential built up in the Na+ ions will be used by many different processes i.e. cotransporters, neuronal signaling etc.

22. P-class pumps: Na+/K+ ATPase pump Binding of phosphate from ATP drives conformation change that allows ions to be transported to appropriate sides: → an asparate residue becomes phosphorylated and the energy transfer changes the proteins conformational shape Na+ binding sites switch from high affinity on inside to low affinity on outside to allow for binding of Na+ on inside and release of Na+ ions on outside. K+ binding sites with from high affinity on outside to low affinity on inside for the same reason

23. P-class pumps: Ca 2+ ATPase pump • pumps 2 Ca2+ ions out for every 1 ATP molecule used • Uses ATP to drive Ca 2+ out against a very large concentration gradient • Internal Ca 2+ binding sites have a very high affinity (in order to overcome extremely low Ca2+ concentrations inside cell) • Energy transfer from ATP to the aspartate of the Ca2+ ATPase causes a protein conformational change and Ca2+ transported across membrane • Ca2+ binding sites on outside are low affinity and Ca2+ is released • The transfer of energy from the ATP to the pump triggers a conformational change that moves the protein and allows the translocation of Ca 2+ across the membrane • At the same time the Ca2+ binding sites change from high to low affinity.

24. P-class pumps: Ca 2+ ATPase pump cont. • In muscle cells the Ca2+ ATPase is the major protein found in the membrane of the sacrcoplasmic reticulum (SR) 80% of the protein in the SR is the Ca2+ ATPase • SR is a storage site for Ca2+ that is release to drive muscle contraction • Ca2+ ATPase will remove excess Ca2+ from the cytoplasm and pump it into the lumen of the SR

25. V-class pumps: proton pumps • These pumps transport H+ only • Found in lysosomes, endosomes and plant vacuoles • Transport H+ ions to make the lumen or inside of the lysosome acidic (pH 4.5 - 5.0) • Many of these pumps are paired with Cl- channels to offset the electrical gradient that is produced by pumping H+ across the membrane.

26. V-class pumps: proton pumps • H+ is transported into the lysosome • Cl- flows in to keep a balance • If Cl- doesn't flow in then there is rapid build up of potential (charge) across the membrane which would block the further transport of H+. • This would occur long before the lumen becomes acidic because not that many ions need to be transported to produce the voltage potential

27. Secondary Active Transport • Use energy held in the electrochemical gradient of one molecule to drive another molecules against its gradient • Antiport or exchanger carrier: molecules move in opposite directions • Symport or cotransporter carrier: molecules move in the same direction

28. Secondary Active Transport Uniporter: One molecule. Amino acids, nucleosides,sugars Symporter/cotransporter: movement in the same directions. Na+/glucose cotransporter in the intestine Antiporter/Exchanger: Cl-/HCO3- exchanger in the red blood cell Example of a Cotransporter

29. Membrane Potential and Na+ • Animal cells are more negative on the inside than on the outside (~ -80 to -70 mV) • Mostly due to K+ ions (inside > outside) created via Na+ /K+ pump, K+ leak channels and anions inside the cell (proteins etc) • Remember K+ will move down its concentration gradient • Nernst potential for K+ is - 80 to -70 mV. Why is this important??? • Transport of Na+ down the chemical gradient and the electrical gradient. Makes Na+ a powerful co-transporter! Favors movement of Na+ into the cell

30. Membrane Potential and Co-transporters Na+/glucose co-transporter • Used by cells in the intestine to transport glucose against a large concentration gradient • This is a symporter: both in the same direction • ΔG for 2 Na+ is -6 kcal/mol

31. Membrane Potential and Co-transporters 3 Na+/Ca2+ antiporter • Important in muscle cells • Maintains the low intracellular concentration of Ca2+ • Plays a role in cardiac muscle • [Ca2+]i = 0.0002 mM and [Ca2+]o = 2 mM • So ΔG = RTln (2/0.0002) = 5.5 kcal.mol ΔG = zFEm = 2(23062)(0.070Volts) = 3.3 kcal/mol Total = 8.8 kcal/mol ►So must transport 3 Na+ in for 1 Ca2+ out

32. Co-transporters HCO3-/Cl- antiporter • Regulate pH • Carbon dioxide from respiration: • CO2 + H2O ↔ H2CO3↔ H+ + HCO3- in the presence of carbonic anhydrase (enzyme) • Note: ~80% of the CO2 in blood is transported as HCO3-. • This is generated by red blood cells (RBC) • RBC have a protein (AE1) and this is the HCO3-/Cl- antiporter • Pumps 1 X 109 HCO3- every 10 msec. • Clears the CO2 and Cl- transport ensures that there isn't a build up of electrical potential

33. Membrane Potential and Co-transporters HCO3-/Cl- antiporter

34. Co-transporters Other transporters that regulate pH Na+/H+ antiporter: Remove excess H+ when cells become acidic Na+HCO3-/Cl- co-transporter: HCO3- is brought into the cell to neutralize H+ in the cytosol: HCO3- + H+↔ H2O + CO2 in the presence of carbonic anhydrase. Driven by Na+: Couples the influx of HCO3- and Na+ to an efflux of Cl-

35. Co-transporters Exchangers are regulated by internal pH and increase their activity as the pH in the cytosol falls