Chapter 9

1. Chapter 9 Membranes and Membrane Transport Biochemistry by Reginald Garrett and Charles Grisham

2. Essential Question • What are the properties and characteristics of biological membranes that account for their broad influence on cellular processes and transport?

3. Outline • What Are the Chemical and Physical Properties of Membranes? • What Is the Structure and Chemistry of Membrane Proteins? • How Does Transport Occur Across Biological Membranes? • What Is Passive Diffusion? • How Does Facilitated Diffusion Occur? • How Does Energy Input Drive Active Transport Processes? • How Are Certain Transport Processes Driven by Light Energy? • How Are Amino Acid and Sugar Transport Driven by Ion Gradients? • How Are Specialized Membrane Pores Formed by Toxins? • What Is the Structure and Function of Ionophore Antibiotics?

4. 9.1 – What Are the Chemical and Physical Properties of Membranes? Structures with many cell functions • Barrier to toxic molecules • Help accumulate nutrients • Carry out energy transduction • Facilitate cell motion • Assist in reproduction • Modulate signal transduction • Mediate cell-cell interactions

5. Lipids Form Ordered Structures Spontaneously in Water Hydrophobic interactions all! • Very few lipids exists as monomers. • Monolayers arrange lipid tails in the air! • Micelles bury the nonpolar tails in the center of a spherical structure. • Micelles reverse in nonpolar solvents.

6. Lipids Form Ordered Structures Spontaneously in Water Hydrophobic interactions all! • Lipid bilayers can form in several ways. • unilamellar vesicles (liposomes) • multilamellar vesicles (Bangosomes, from Bangham) • The nature and integrity of these vesicle structures are very much dependent on the lipid composition.

7. The Fluid Mosaic Model Describes Membrane Dynamics S. J. Singer and G. L. Nicolson • The phospholipid bilayer is a fluid matrix. • The bilayer is a two-dimensional solvent. • Lipids and proteins can undergo rotational and lateral movement. • Two classes of proteins: • peripheral proteins (extrinsic proteins): associate with membrane by ionic interactions and H-bonds. • integral proteins (intrinsic proteins): by hydrophobic interactions, can only be dissociated by detergents or organic solvents.

8. Membrane bilayer thickness: 5 nm by X-ray • The electron density of interiors of the membranes (the hydrocarbon tails) are low, while that of the outside edges (the polar head groups) of the same membrane are high. • Hydrocarbon chain orientation in the bilayer: The tails tilt and bend and adopt a variety of orientations. The portions of a lipid chain lie nearly perpendicular near the membrane surface, while the chains toward the middle of the bilayer are not ordered.

9. Motion in the Bilayer Lipid chains can bend, tilt and rotate. • Lipids and proteins can migrate ("diffuse") in the bilayer. • Frye and Edidin proved that integral membrane proteins can move laterally, using fluorescent-labelled antibodies.(Figure 9.7) • Lipid diffusion has been demonstrated by NMR and EPR (electron paramagnetic resonance) and also by fluorescence measurements. • How fast? A few m/min, or <10 nm/sec for integral proteins, which anchored to the cytoskeleton to maintain cell’s shape. Several m/sec for lateral movement of lipids. Transverse movement of lipids or proteins are much slower (in days).

10. Membranes are Asymmetric Structures • Lateral Asymmetry of Lipids: • Lipids can cluster in the plane of the membrane - they are not uniformly distributed. • Ex. Ca+2 induced clustering (phase separation) of PS, PE, and PC. (Fig 9.8) --- regulate the activity of membrane-bound enzymes. • Intercalation of cholesterol can affect the function of membrane proteins and enzymes. • Distribution of lipids can be affected by proteins --- preference of saturated FA chains or head groups.

11. Lateral Asymmetry of Proteins: • Proteins can associate and cluster in the plane of the membrane - they are not uniformly distributed in many cases. • Some proteins form multisubunit complexes to perform specific functions. Ex. The “purple patches” bacteriorodopsinprotein (Fig 9.9).

12. Membranes are Asymmetric Structures • The two sides of a membrane bilayer are different. Ex. Membrane transport is for one direction. Hormone interactions and immunological reactions are at the outside surface of cells. • Transverse asymmetry of proteins • Mark Bretscher showed that N-terminus of glycophorin is extracellular whereas C-terminus is intracellular.(Fig 9.14) • Transverse asymmetry of lipids • In most cell membranes, the composition of the outer monolayer is quite different from that of the inner monolayer.(Fig 9.10) • The carbohydrate groups of glycolipids and glycoproteins always face outside of the membrane.

13. Transverse asymmetry • Asymmetric lipid distributions led to difference in total charge and the membrane potential on the inner and outer surfaces. The membrane potential modulates the activity of certain ion channels and membrane proteins. • Lipid asymmetry due to two processes: • asymmetric synthesis of phospholipid, glycolipid, and cholesterol in ER and Golgi system and flow by lipid transfer proteins. • (B) energy-dependent transport --- flippases (Fig 9.11) reduce t1/2 from 10 days to a few minutes, passively or in an ATP-dependent manner.

14. Flippases • A relatively new discovery! • Lipids can be moved from one monolayer to the other by flippase proteins. • Some flippases operate passively and do not require an energy source. • Other flippases appear to operate actively and require the energy of hydrolysis of ATP. • Active flippases can generate membrane asymmetries.

15. Membranes Undergo Phase Transitions • the "melting" of membrane lipids • Below a certain transition temperature, membrane lipids are rigid and tightly packed. • Above the transition temperature, lipids are more flexible and mobile. • The transition temperature is characteristic of the lipids in the membrane. • Only pure lipid systems give sharp, well-defined transition temperatures.

16. Membrane phase transitions • Radical change in physical state occurring within narrow range of transition (or melting) temperature. • Below Tm: Lipids close-pack: Lose lateral mobility and rotational mobility of fatty acid chains. • Consequences: Membrane thickens and decreases surface area. • Characteristics: (1) endothermic • (2) Tm increases with chain length and degree of saturation and is influenced by nature of head group. • (3) Pure phospholipid bilayers show narrow temperature range --- cooperative phase change. • (4) Native membranes show broad transition influenced by protein and lipid composition. • (5) Pretransition (5-15 C below Tm) involves tilting. • (6) Increased volume (7) sensitive to interacting cations and lipid-soluble agents. Cells adjust lipid composition of their membranes to maintain fluidity as the environments change.

17. 9.2 – What Is the Structure and Chemistry of Membrane Proteins? • Functions: transport, receptor, etc. • Singer and Nicolson defined two classes. • Integral (intrinsic) proteins • Peripheral (extrinsic) proteins • lipid-anchored proteins: covalent linkage

18. Peripheral Proteins • Peripheral proteins are not strongly bound to the membrane. • They can be dissociated with mild detergent treatment or with high salt concentrations.

19. Integral Membrane Proteins Are Firmly Anchored in the Membrane • Integral proteins are strongly imbedded in the bilayer, by -helices or -sheets due to neutralization of H-bonds of N-H and C=O peptide backbone. • They can only be removed from the membrane by denaturing the membrane (organic solventsor strong detergents). • Often transmembrane, but not necessarily. • Glycophorin (Fig 9.14) and bacteriorhodopsin (Fig 9.15) are examples.

20. Glyophorin A single-transmembrane-segment protein • One transmembrane segment with globular domains on either end. • Transmembrane segment is -helical and consists of 19 hydrophobic amino acids. • Extracellular portion contains oligosaccharides and these constitute the ABO and MN blood group determinants, which also serve as receptor for influenza virus. • 40% protein and 60% carbohydrate by weight.

21. Bacteriorhodopsin A 7-transmembrane-segment (7-TMS) protein • Found in purple patches of Halobacterium halobium. • Consists of 7 transmembrane helical segments with short loops that interconnect the helices back and forth across the membrane. Very little exposed to aqueous milieu. • Note the symmetry of packing of bR (see Figure 9.15) • bR is a light-driven proton pump!

22. Porins --- a -sheet motif for membrane proteins Found both in Gram-negative bacteria and in mitochondrial outer membrane • Porins are pore-forming proteins - 30-50 kD • General or specific - exclusion limits 600-6,000 • Most arrange in membrane as trimers. • High homology between various porins. • Porin from E. colihas 18-stranded beta barrel that traverses the membrane to form the pore (with eyelet!).

23. Figure 9.16-17. The three-dimensional structure (left) and the arrangement of the peptide chain (right) of maltoporin from E. coli. Maltoporin participates in the entry of maltose and maltodextrins into E. coli. Polar and nonpolar residues alternate along the -strands, with polar residues facing the cavity of the barrel and nonpolar residues facing out, interacting with the hydrophobic lipid milieu of the membrane.

24. Why Beta Sheets? • Genetic economy • -helix requires 21-25 residues per transmembrane strand. • -strand requires only 9-11 residues per transmembrane strand. • Thus, with -strands , a given amount of genetic material can make a larger number of transmembrane segments. • -strands can present alternate polar and nonpolar residues along the -strands. Polar residues face the hydrophilic cavity of the barrel and nonpolar residues face the hydrophobic lipid bilayer.

25. Treating allergies at the cell membrane Antihistamine drugs bind tightly to histamine H1 receptors, without eliciting the effect of histamine. p.280

26. Lipid-Anchored Membrane Proteins Are Switching Devices A class of membrane proteins covalently bound to lipid molecules • The activity of the protein can be modulated by attachment of the lipid. • The reversible attachment of the lipid can serve as a “switching device” for altering the affinity of a protein to the membrane and controlling signal transduction pathways. • Four types have been found: • Amide-linked myristoyl anchors • Thioester-linked fatty acyl anchors • Thioether-linked prenyl anchors • Glycosyl phosphatidylinositol anchors

27. Amide-Linked Myristoyl Anchors • Always myristic acid(14:0 fatty acid) • Always N-terminal • Always a Gly residue that links • Examples: cAMP-dependent protein kinase, pp60src tyrosine kinase, calcineurin B, alpha subunits of G proteins, gag protein of HIV-1

28. Thioester-linked Acyl Anchors • Broader specificity for lipids - myristate, palmitate, stearate, oleate all found. • Broader specificity for amino acid links - Cys, Ser, Thr all found. • Examples: G-protein-coupled receptors, surface glycoproteins of some viruses, transferrin receptor triggers and signals.

29. Thioether-linked Prenyl Anchors • Prenylation refers to linking of "isoprene"-based groups. • Always Cys of CAAX (C=Cys, A=Aliphatic, X=any residue) • Isoprene groups include farnesyl (15-carbon, three double bond) and geranylgeranyl (20-carbon, four double bond) groups • Examples: yeast mating factors, p21ras and nuclear lamins

30. Glycosyl Phosphatidylinositol Anchors • GPI anchors are more elaborate than others. • Always attached to a C-terminal residue. • Ethanolamine link to an oligosaccharide linked in turn to inositol of PI. • See Figure 9.20 • Examples: surface antigens, adhesion molecules, cell surface hydrolases.

31. 9.3 How does transport Occur Across Biological Membranes? • Cells must exchange materials with their environment. Bring nutrients in and send waste products out of cellular membrane and organelles. • Concentration gradients must be maintained. • Na+ and K+ gradients mediate transmission of nerve impulses and normal functions of the brain, heart, kidneys, and liver. Ca+2 controls muscle contractions and hormonal responses. • High [H+] in mucosal membrane of the stomach. • Need transport proteins for water-soluble molecules. • Membrane transport: 3 types • Passive diffusion • Facilitated diffusion • Active transport: an energy driven process

32. 9.4 – What is Passive Diffusion? Thermodynamically favorable. No special proteins needed. • Entropically driven process: Moleculessimply moves down its concentration gradient - from high [C] to low [C]. • ∆G = RTln([C2]/[C1]) for uncharged molecule • ∆G = RTln([C2]/[C1]) + ZF∆ for charged molecules R = 8.32 J/K·mol, T = K, Z = charge, F = 96,485 J/V·mol, ∆ = electrical potential Rate depends on concentration gradient and lipid solubility.

33. 9.5 – How Does Facilitated Diffusion Occur? G negative, but proteins assist • Solutes only move in the thermodynamically favored direction. • But proteins may "facilitate" transport, increasing the rates of transport. • Understand plots in Figure 9.23 • Two important distinguishing features: • Solute flows only in the entropically favored direction. • Involves integral membrane protein • Rate depends on concentration but is saturatable. • Specificity and affinity due to protein/transported molecule interaction. Examples: • Glucose transporter: RBC band 4.5: 55 kD protein functions as trimer • Anion transport system: RBC band 3: 95 kD protein in Cl-, HCO3- exchange

34. Vmax Km Figure 13.7, p.413 Figure 9.23 Passive diffusion and facilitated diffusion may be distinguished graphically. The plots for facilitated diffusion are similar to plots of enzyme-catalyzed processes (Chapter 13) and they display saturation behavior.

35. 9.6 – How Does Energy Input Drive Active Transport Processes? Energy input drives transport • Some transport must occur such that solutes flow against thermodynamic potential. • Energy input drives transport. • Energy source and transport machinery are "coupled“. • Energy source may be ATP, light or a concentration gradient. • Active transport: Energy driven process • Primary active transport: Energy sources • ATP hydrolysis (most common) • Light energy • Secondary active transport (Energy is ion gradient formed by some other process.) • Electrogenic transport: Active transport of ions and net charge transport both occur.

36. The Sodium Pump Na+,K+-ATPase • Large protein - 120 kD and 35 kD subunits • Maintains intracellular [Na+] low (10 mM) and [K+] high (100 mM). • Crucial for all organs, but especially for neural tissue and the brain. • 3 Na+ out, 2 K+ in per ATP hydrolyzed: Electrogenic. • Ouabain: Cardiac glycoside that inhibits sodium pump. • Alpha subunit has 10 transmembrane helices with large cytoplasmic domain.

37. Na+,K+ Transport • ATP hydrolysis occurs via an E-P intermediate. • Mechanism involves two enzyme conformations known as E1 and E2. Binding of Na+ ions to E1 is followed by phosphorylation and release of ADP. E2-P has low affinity for Na+ and high affinity for K+. Na+ ions are transported and released and K+ ions are bound before dephosphorylation of the enzyme. • Cardiac glycosides inhibit by binding to the extracellular surface of Na+,K+-ATPase in the E2-P state.

38. E2P: low affinity for Na+ high affinity for K+ Figure 9.29A mechanism for Na+,K+-ATPase . The model assumes two principal conformations, E1and E2. Binding of Na+ ions to E1 is followed by phosphorylation and release of ADP. Na+ ions are transported and released and K+ ions are bound before dephosphorylation of the enzyme. Transport and release of K+ ions complete the cycle.

39. Na+,K+ Transport • Hypertension involves apparent inhibition of sodium pump. Inhibition in cells lining blood vessel walls results in Na+,Ca+2 accumulation and narrowing the vessels. • Studies show this inhibitor to be ouabain!

40. Calcium Transport: Ca2+-ATPase A process akin to Na+,K+ transport • Calcium levels in resting muscle cytoplasm are maintained low by Ca2+-ATPase - a Ca2+ pump. • In muscles, [Ca+2]= 0.1 M in resting state; [Ca+2]= 10 M during contraction. • Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the -subunit of Na,K-ATPase. • Aspartyl phosphate E-P intermediate is at Asp-351 and Ca2+-pump also fits the E1-E2 model.

41. Ca2+-ATPase Fig. 9-31, p.292 FIGURE 9.31 The structure of Ca2+-ATPase. The transmembrane (M) domain is shown in red, the nucleotide (N) domain is shown in blue, the phosphorylation (P) domain is purple, and the actuator (A) domain is green. The phosphorylation site, Asp- 351, is yellow. Two Ca2+ ions in the transmembrane site are green.

42. Cardiac Glycosides: Potent Drugs from Ancient Times 夾竹桃 Stimulation of the Na+-Ca+2 exchanger increased intracellular [Ca+2] and stimulate muscle contraction to benefit patients with heart problems. p.293

43. The Gastric H+,K+-ATPase The enzyme that keeps the stomach at pH 0.8 • The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4 • H+,K+-ATPase pumps H+from the mucosal cells into the stomach in exchange of K+, to maintain a pH difference of 6.6 across a single plasma membrane! ---a electronically neutral process. • K+ is pumped back together with Cl-. • Net transport of HCl into the interior of stomach.

44. The Gastric H+,K+-ATPase • This is the largest concentration gradient across a membrane in eukaryotic organisms! • 1 H+ out, 1 K+ in per ATP hydrolyzed. • Gastric enzyme: ∆pH largest gradient known. • H+,K+-ATPase is similar in many respects to Na+,K+-ATPase and ER Ca+2-ATPase, forming E-P intermediate.

45. Osteoclast Proton Pumps How your body takes your bones apart? • 5% bone material undergoes ongoing remodeling at any given time. • osteoclasts tear down bone tissue • osteoblasts build it back up • Osteoclasts function by secreting acid into the space between the osteoclast membrane and the bone surface.Acid (pH 4) dissolves the Ca-phosphate (hydroxyapatitie) matrix of the bone. • An ATP-driven proton pump in the membrane does this!

46. The ATPase that Transport peptides and Drugs • Animal cells have a transport system that is designed to recognize foreign organic molecules. • This organic molecule pump recognizes a broad variety of molecules and transports them out of the cell using the hydrolytic energy of ATP.

47. The MultiDrug Resistance ATPase Also known as the P-glycoprotein • MDR ATPase is a member of a "superfamily" of genes/proteins that appear to have arisen as a "tandem repeat". • It recognizes, binds, and transports a broad group of structurally diverse molecules with unknown mechanism. • MDR ATPase defeats efforts of chemotherapy.

48. 9.7 – How Are Certain Transport Processes Driven by Light Energy? the Bacteriorhodopsin story • Halobacterium halobium, the salt-loving bacterium (optimum [NaCl]= 4.3M), carries out normal respiration if O2 and substrates are plentiful. • But when substrates are lacking, it can survive by using bacteriorhodopsin and halorhodopsin to capture light energy. • Purple patches of H. halobium are 75% bR (the only protein) and 25% lipid - a "2D crystal" of bR - ideal for structural studies --- 7 transmembrane helical segments (Figure 9.15).

49. Figure 9.36 The Schiff base linkage between the retinal chromophore and Lys216 gives rise to the purple color. Figure 17.36 p. 571

50. Fig. 20-28, p. 662 Figure 9.37 The reaction cycle of bacteriorhodopsin. The intermediate states are indicated by letters, with subscripts to indicate the absorption maxima of the states. Also indicated for each state is the configuration of the retinal chromophore (all-trans or 13-cis) and the protonation state of the Schiff base (C=N: or C=N+H).