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MEMBRANE TRANSPORT

MEMBRANE TRANSPORT. Bob Mercer rmercer@wustl.edu September 19, 2013. POLYCYSTIC RENAL DISEASE. 1 in 500 autopsies 1 in 3000 hospital admissions Accounts for ≈10% of end-stage renal failure Autosomal dominant inheritance. WILSON’S DISEASE.

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MEMBRANE TRANSPORT

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  1. MEMBRANE TRANSPORT Bob Mercer rmercer@wustl.edu September 19, 2013

  2. POLYCYSTIC RENAL DISEASE 1 in 500 autopsies 1 in 3000 hospital admissions Accounts for ≈10% of end-stage renal failure Autosomal dominant inheritance

  3. WILSON’S DISEASE 1 in 100 individuals carry mutation in ATP7B gene (Cu-ATPase) 1-4 per 100,000 people Autosomal recessive inheritance Neurological or psychiatric symptoms Liver disease Kaysar-Fleischer (KF) ring

  4. CYSTIC FIBROSIS 1/2000 births in white Americans Median age for survival late 30s Autosomal recessive inheritance

  5. COMPARISON OF ION CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN CELL Intracellular Extracellular Concentration Concentration Component (mM) (mM) Cations Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4) Anions Cl 5-15 110 Because the cell is electrically neutral the large deficit in intracellular anions reflects the fact that most cellular constituents are negatively charged. The concentrations for Mg and Ca are given for free ions.

  6. A Cross between Human Beings and Plants . . .SCIENTISTS ON VERGE OF CREATING PLANT PEOPLE . . .Bizarre Creatures Could do Anything You Want Tuesday, July 1, 1980

  7. Brain water (g/100 g dry wt)

  8. Simple Diffusion • Flux is proportional to external concentration • Flux never saturates Flux [S]o

  9. PROTEIN MEDIATED MEMBRANE TRANSPORT • PRIMARY ACTIVE • SECONDARY ACTIVE TRANSPORT • FACILITATED DIFFUSION • ENDOCYTOSIS/TRANSCYTOSIS

  10. Membrane Flux (moles of solute/sec) • Simple Diffusion • Carrier Mediated Transport • Facilitated Diffusion • Primary Active Transport • Secondary Active Transport • Ion Channels

  11. TRANSPORT OF MOLECULES THROUGH MEMBRANES

  12. CARRIER MEDIATED TRANSPORT

  13. Membrane Potential Review • The lipid bilayer is impermeable to ions and acts like an electrical capacitor. • Cells express ion channels, as well as pumps and exchangers, to equalize internal and external osmolarity. • Cells are permeable to K and Cl but nearly impermeable to Na. • Ions that are permeable will flow toward electrochemical equilibrium as given by the Nernst Equation. Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C • The Goldman-Hodgkin-Katz equation is used to calculate the steady-state resting potential in cells with significant relative permeability to sodium.

  14. Structure of a Potassium Channel Doyle et al., 1998

  15. Carrier-Mediated Transport • Higher flux than predicted by solute permeability • Flux saturates • Binding is selective (D- versus L-forms) • Competition • Kinetics: [S]o << KmM a [S] [S]o = KmM = Mmax / 2 [S]o >> KmM = Mmax Mmax Flux 0.5 Km [S]o

  16. MEMBRANE ION TRANSPORT PROTEINS

  17. k + k - So + Co SCo Si S = Solute C = Carrier Transport Kinetics dSCo/dt = k+ [S]o [C]o – k- [SC]o = 0 at equilibrium Þ k+ [S]o [C]o = k- [SC]o k-/k+ = ([S]o [C]o)/[SC]o = KmÞ [SC]o = ([S]o [C]o)/Km Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o) M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o)

  18. Co Ci So Si SCo SCi Mnet = Min – Mout = Mmax ) ( 1 1 1 + Km / [S]o 1 + Km / [S]i - Reversible Transport

  19. Facilitated Diffusion • Uses bidirectional, symmetric carrier proteins • Flux is always in the directions you expect for simple diffusion • Binding is equivalent on each side of the membrane Examples include: Glucose Transporters (GLUT); Anion Exchanger; Organic Anion Transporters; Urea Transporters; Monocarboxylate (lactate) Transporters (MCTs); Amino Acid Transporters; Zn Transporters (ZIP)

  20. Facilitated Diffusion • Uses bidirectional, symmetric carrier proteins • Flux is always in the directions you expect for simple diffusion • Binding is equivalent on each side of the membrane

  21. Facilitated Diffusion: Band 3/AE1

  22. Facilitated Diffusion: Band 3/AE1

  23. Cytoskeletal/AE1 Interactions

  24. Primary Active Transport: Driven by ATP • Class P – all have a phosphorylated intermediate • Na,K-ATPase H,K-ATPase • Ca-ATPase • Cu-ATPase • H-ATPase • bacterial K-ATPase • Phospholipid Flippase • Class V • H+ transport for intracellular organelles • Class F • Synthesize ATP in mitochondria

  25. 3 Na ATP ADP + Pi 2 K Primary Active Transport:Na,K-ATPase • 3 Na outward / 2 K inward / 1 ATP • Km values: Nain = 20 mM Kout = 2 mM • Inhibited by digitalis and ouabain • Palytoxin “opens” ion channel • 2 subunits, beta and alpha (the pump) • Two major conformations E1 & E2 • Turnover = 300 Na+ / sec / pump site @ 37 °C

  26. Na,K-ATPase Reaction Scheme

  27. Membrane Transport and Cellular Functions that Depend on the Na,K-ATPase

  28. Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms

  29. The Na,K-ATPase As a Receptor For Signal Transduction

  30. SR Ca-ATPase

  31. FoF1 ATPase

  32. Nat Commun. 2012 February 21; 3: 687

  33. Experimental Evidence for Rotation

  34. Secondary Active Transport • Energy stored in the Na+ (or H +) gradient is used to power the transport of a variety of solutes glucose, amino acids, ions and other molecules are pumped in (cotransport) Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+ (countertransport) • These transport proteins do not hydrolyze ATP directly; but they work at the expense of the ion gradient which must be maintained by an ATPase

  35. Secondary Active Transport • In humans over 40 families of Na coupled transporters Examples include: Na+/H+ exchanger; Na+/Ca2+ exchanger; Na+/aspartate cotransporter; Na+/amino acid cotransporter; Na+/glucose cotransporter; Na+/urea cotransporter; Na+/PO4; cotransporter; (H+/Na+)/Zn2+ exchanger (ZnT)

  36. Secondary Active Transport • Energy stored in the Na+ gradient is used to power the transport of a variety of solutes glucose, amino acids and other molecules are pumped in (cotransport) Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+ (countertransport) • These transport proteins do not hydrolyze ATP directly; but they work at the expense of the Na+ gradient which must be maintained by the Na,K-ATPase

  37. Energy available from ATP H2O ATP ADP + Pi DG = Gproducts – G reactants Chemical Energy (G) = RT ln [C] DG = DG° + 2.3 RT (log ([ADP] [Pi]) – log [ATP]) 2.3 RT = 5.6 kiloJoules / mole @ 20° C DG° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions”

  38. DG = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi] Energy Depends on Substrate Concentrations • The energy available per molecule of ATP depends on: • [ATP] @ 4mM, [ADP] @ 400 µM, [Pi] @ 2 mM • Þ per mole of ATP hydrolyzed: • DG = -30 kJ – 5.6 kJ * log 4 x 10-3 • 2 x 10-3 * 4 x 10-4 • = -30 kJ - 21 kJ = -51 kiloJoules per mole of ATP • Converting to approximately -530 meV/molecule of ATP

  39. Energy in the Sodium Gradient Consider Na+ movement from outside to inside: G = Gproducts – Greactants = Ginside – Goutside Gtotal = Gelectrical + Gchemical Conditions for our sample calculation: Vm = -60 mV [Na+]out = 140 mM [Na+]in = 14 mM and 2.3 RT = 60 meV / molecule

  40. Energy in the Na Gradient: Electrical Term • Gelectrical = e mVin – e mVout • = +1e -60 mV – (+1e) 0 mV • = -60 meV • negative sign means energy is released moving from outside to inside • 60 meV is the energy required to move a charged ion (z=1) up a voltage gradient of 60 mV (assuming zero concentration gradient) * * * *

  41. Energy in the Na Gradient: Chemical Term • DGchemical = 2.3 RT (log [Na+]in – log [Na+]out) • = 60 meV * (-1) • = -60 meV • negative sign means energy is released moving from outside to inside • 60 meV is the energy required to move a molecule up a 10 fold concentration gradient (true for an uncharged molecule or for a charged molecule when there is no voltage gradient)

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