Biological Membranes & Transport

Life without membranes? Life without cells? 1. What are membranes made of? 2. Membranes, static or dynamic? 3. Transport across membranes?

View The Inner Life of the Cell video8 minute version: http://www.youtube.com/watch?v=yKW4F0Nu-UY

partial script (w/thumbnails): http://sparkleberrysprings.com/innerlifeofcell.html

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I. Membrane Composition & Architecture

A. Each membrane type: characteristic lipids,proteins:

1. Protein-lipid ratios, Table 11-1, p. 386

2. Different membranes-different lipids, Fig. 11-2

3. Different membranes-different proteins. a. rhodopsin (in our eyes & some bacteria)b. acetylcholine esterase (Where? )

But some tissue specificity2

B. All biological membranes share fundamentalproperties

1. Fluid mosaic model (lipid is in liquid state)

2. Phospholipids form a bilayer (solvent of membranes)that is quantitatively asymmetric.

3. Membrane proteins are embedded in or stuck to thesurface of the lipid bilayer.

4. Orientation of membrane proteins is qualitativelyasymmetric. Summary in Fig. 11-3. (Wavy?)View animation?

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C. Lipid bilayer is basic structural element ofmembranes & its formation is spontaneous (onceyou have the parts). Fig. 11-4Types of aggregates:

1. Micelles (Note curvature)2. Bilayers3. Vesicles (liposomes) (Note curvature)4. Back to asymmetric distribution of lipids, Fig. 11-5.

Comment re micelles, vesicles, and bilayers.

The dominant form that is present is determined by thestructure of the lipid.

Solubility vs. critical micelle concentration (cmc).4

Erythrocyte plasma membranes (the archetypalmembrane system): quantitatively asymmetric forinner/outer lipid mono-layer composition:

D. 3 types of membrane protein differ in theirassociation w/ the membrane.

1. See Fig. 11-7 re. peripheral, integral, amphitropic 2. Interactions responsible for holding in/on membrane:

a) integral proteins b) peripheral proteins c) amphitropic Amphitropic distribution (membrane vs. cytosol) changes

through time (usually in a regulated manner).

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Fig. 11-7

E. Many membrane proteins span the lipid bilayer.

1. What thermodynamic problem is associated withproteins spanning (crossing?) the lipid bilayer?

2. Glycophorin (Fig. 11-8): well characterized example

Fig 11-8. Glycophorin

Lot’s of interesting details here.

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Aside: I only semi-understand the book’s comment re.“...residues 64 to 74 has some hydrophobic residues and probablypenetrates the outer face of the lipid bilayer as shown.”

F. Integral proteins are held in the membrane byhydrophobic interactions with lipids. (Fig. 11-8-10)1. Sequence was the initial clue for this. Topologies:

Figure 11-9 (Error: labels Type IV & Type V arereversed?)

2. 3-D structure examined by X-ray methods:a) Bacteriorhodopsin: 7 spans! Fig. 11-10, p. 391b) Lipids coat the hydrophobic regions of aquaporin

and F0 Na+-ATPase. Fig. 11-11

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G. Topology of integral membrane proteins can bepredicted from their sequence. 1. Most common ways to cross membrane:

a) α-helixb) β-sheet (Fig. 11-14)

2. How do you find likely membrane spanningregions? Hydropathy plots for glycophorin andbacteriorhodopsin. Fig. 11-12.

Positive inside rule: Arg-Arg-Leu-Ile-Lys-Lys glycophorin

Remember hydropathy values from Chapter 3? (Seenext page.)

How are hydropathy values determined?

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Values come from Table 3-1 on p. 77.amino hydro  pathyacid value    Gly  ‐0.4Ala 1.8Pro ‐1.6Val 4.2Leu 3.8Ile 4.5Met 1.9Phe 2.8Tyr ‐1.3Trp ‐0.9Ser ‐0.8Thr ‐0.7Cys 2.5Asn ‐3.5Gln ‐3.5Lys ‐3.9His ‐3.2Arg ‐4.5Asp ‐3.5Glu ‐3.5

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Fig. 11-12 Comment on windows?

Back to Table 3-1?

3. Visual representation of composition in Fig. 11-13. Think about extractions you performed in organicchemistry lab.

Spanning the membrane with β-sheets (barrels?)See also pdb 2omf

H. Covalently linked lipids can anchor membraneproteins.

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1. There’s more than one way to stick to a membrane.

2. Summary: Fig. 11-15.a) Different residue linkages to proteinsb) Different lipid components as anchorsc) Different surface distributions of anchors and proteins

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II. Membrane Dynamics

The liquid state of membrane lipids implies that themolecules associated with the membrane will be quitemobile (dynamic) unless something ties them down.

A. Acyl groups in the bilayer interior are orderedto varying degrees. States:1. Gel (paracrystalline), highly ordered, @ low temp2. Liquid ordered, intermediate order, @ middle temps3. Fluid, liquid disordered, lots of motion, @ high temp

See Fig. 11-16, p. 395 (density change?)View animation?

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4. Some cells can alter their membrane lipidcomposition to maintain constant fluidity:

Note “Ratio” row.

B. Transbilayer lipid movement requires catalysis.

1. If a process is slow (uncatalyzed, this one is) whatdoes that mean energetically (thermodynamically)?

Contrast with diffusion within a monolayer:

2. Flippases, floppases, etc. increase the rate of transitbetween the inner and outer layers of a bilayer:

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Extracellular monolayer PS signals.........?

C. Lipids & proteins diffuse laterally in thebilayer. Evidence:

1. Fluorescence recovery after photobleaching (FRAP). Fig. 11-18

An elegant experiment.View animation?

2. Single particle tracking: restrictions on movement. Fig. 11-19: 2,250 frames.

Time resolution = 25 μs

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3. Protein based lattice work (ankyrin and spectrin, Fig11-20) may represent some of the fencing inerythrocytes. Inner Life of the Cell video?

D. Sphingolipids & cholesterol cluster together inmembrane rafts. Solubility, functional significance?

1. Cholesterol/sphingolipid microdomains are thicker &more ordered than neighboring microdomains; rafts

2. These rafts are enriched in 2 classes of integralmembrane proteins.

a) anchored w/ 2 fatty acids covalently linked to Cysb) GPI-anchored proteins (see Fig. 11-15 to review both)

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3. Raft details:a) diameter = 50 nmb) This size translates to a few thousand lipids and perhaps

10 to 50 membrane proteins (only a few protein types?). Functional significance?

c) Up to 50% of a plasma membrane surface can be rafts. (A crowded ocean?)

d) Lipids/proteins constantly move in & out of rafts.

4. Some rafts contain caveolin (see membraneattachment pattern in Fig. 11-21). Caveolin dimersforce inward curvature of the membrane (Fig. 11-22). Caveolin rafts usually involve both monolayers. This is uncommon for rafts. Note violation of the“can’t go half-way in and come back out” rule.

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E. Membrane curvature & Fusion are central tomany biological processes. When did you 1st become you? List of processes, Fig. 11-23

Increased curvature due to:1. Charge density issues

Fig. 11-242. Individual protein action3. Protein scaffolding interactions (BAR

domains). 4. Some proteins function to bring membranes

into close contact so fusion can occur Fig. 11-25v- & t-SNARE zipper mechanism in synaptic vesicle fusion.

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F. Integral membrane proteins are involved insurface adhesion, signaling, and other cellularprocesses. Note Ca2+ binding domains.1. What holds our cells together?

a) integrin (αβ dimers) β subunit genetic disease.b) cadherinc) N-CAM (Ca2+ independent interactions)d) selectin

2. Many of these perform other critical functions (bloodclotting, catalysis, transport, etc.)

Comment: anthrax toxin function/endosome pH changes.

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III. Solute Transport across Membranes

Some general comments (A, B & E), then manyexamples. Transport can be broadly characterized aspassive (“with” an electrochemical gradient) or active(“against” said gradient). See 1st summary figure, nextpage.

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A. Passive transport: membrane proteins facilitate

1. Q: Why are specialized transport systems selectivelyadvantageous?

2. The term “transport” implies change (in location). Recall from your previous chemistry that changerequires a driving force.

3. “Passive transport” describes transport driven by anexisting electrochemical gradient. Electrochemical?(see next pages)

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a) Chemical gradient: [S]outside … [S]inside (Really, activity.)Boltzmann program?

b) Electrical gradient: chargeoutside … chargeinside Spend a fewmoments on Fig. 11-27. How to generate the left handconditions in 11-27 (a) & (b)?

4. Four types of passive transport: Back to Fig. 11-26. (6- noon).

a) All four are biologically important.i) Simple diffusionii) Ionophoresiii) Ion channeliv) Fascilitated diffusion (down electro &/or chemical gradient)

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b) What sorts of compounds can do simple diffusion (atreasonable rates)?

c) What sorts of compounds can’t do simple diffusion?

5. Thermodynamic picture of transport, Fig. 11-28. Look familiar? What is ΔGE, as shown?

B. Transporters & Ion Channels are different.

1. Outcome of genomic analysis: lots of sequence info.

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a) This allows us to establish links between genes. (Biochemical/genetic basis? Gene duplication!)

b) Can you provide examples from earlier chapters?

c) Do proteins with similar sequences always have similar(identical?) functions?

d) Do proteins with different sequences ever have similarfunctions?

2. Many human transporter genes ($1000) exist. Wetherefore need to organize the way we view them. There are (many?) different ways to categorize, but2 categories: Channels & Transporters. Fig 11-29

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a) Channels generally:i) bind S with less stereospecificity than carriersii) transport S at rates toward diffusion limitsiii) are usually not saturable with respect to S

c) Transporters (pumps) generally: i) bind substrates (S) with high stereospecificityii) transport S at rates well below diffusion limitsiii) are saturable with respect to S (like enzymes)

Fig 11-29 Note: This is a cartoon.

3. Channels & transporters represent two of thebroadest ways to categorize transporter systems.

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4. Structurally, some transporters are clustered α-helices (GLUT1) & some are β-barrels (porins).

5. Some carriers function through an existingelectrochemical gradient; some use active transport.

The remainder of Chapter 11 looks in more detail at anumber (~8) different transport systems (or system types),active transport (generally and through ion gradients), &a specific method to measure ion channel function.

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C. The glucose transporter of erythrocytes(GLUT1) mediates passive transport.

1. GLUT1 does facilitated diffusion (Fig 11-26, noon)

2. Structurally (Fig. 11-30 a):a) A type III integral membrane protein (Fig. 11-9)b) Mr . 45,000c) 12 (long enough to span membrane) hydrophobic runsd) Postulated arrangement in Fig. 11-30 b) & c)

How do the helices fit together? Part of one helix shown in 11-3-b 4 helices shown 11-30c

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3. Kinetics: Glucose by GLUT1 transport is 50,000 xfaster than by simple diffusion through membrane.a) See Fig. 11-31. Look familiar? Kt = Ktransportb) Binding of glucose is (stereo)specific & saturable.c) [glucose]blood . 5 mM (fasting ?)

i) D-glucose Kt = 1.5 mMii) D-mannose Kt = 20 mM, D-galactose Kt = 1.5 mM

d) Postulated mechanism in Fig. 11-32, below.

Why (in vivo) is transport essentially unidirectional?

5. Other glucose transporters (12 known in us, Table 11-3) a) GLUT2 ships glucose out of liver (Kt = 66 mM)b) GLUT4 and diabetes mellitus, Box 11-1

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Box 11-1

D. The Cl!-HCO3! exchanger catalyzes

electroneutral cotransport of anions across theplasma membrane (example of antiport).

1. a.k.a. Anion exchange (AE) protein

2. Ultimate function: increase rate of HCO3! transport

by blood. Rate enhancement . 106.

3. Structural pattern: similar to GLUT1?

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4. Action: Cl! & HCO3! co-transport must occur

a) simultaneously (cotransport) Fig 11-33 & b) in opposite directions (antiport see Fig. 11-34)

Again, many different ways to categorize transportsystems.

5. There are three different Cl!-HCO3! exchanger genes

a) Red blood cells express AE1b) AE2 gene product present in large amounts in liver c) AE3 in plasma membranes of brain, heart, & retina

E. Active transport results in solute movement against aconcentration or electrochemical gradient.1. Two approaches, see Fig. 11-35

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a) 1E: direct energy coupled movement of 1 speciesb) 2E: energy coupling through movement of 2nd species

2. Back to thermo: ΔG = ΔGNE + RT ln ([P]'[S])

Recall ΔG = ΔGNE + RT ln Q from CHM 112?

3. Transported species not ionic: ΔG = RT ln (C2'C1)

a) The variables? ΔG RT

C2 & C1 (Transport region 1 to region 2.)If you understand where ΔGNE went, you are starting to

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get this.

b) What happens if S is an ion?

ΔG = RT ln (C2'C1) + Zö ΔψZö Δψ

Make sure you can do problems like the workedexamples (11-1 & 11-2) with great alacrity.

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F. P-type ATPases undergo phosphorylationduring their catalytic cycles.

1. Cellular outcome re. memebrane potential, [Na+],[K+] Fig. 11-36

2. Mechanism, Fig. 11-37

3. Na+'K+ ATPase function in animals Fig. 11-38

G. V-type & F-type ATPases are ATP-driven H+

pumps.Read on your own.

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H. ABC transporters use ATP to drive the activetransport of a wide variety of substrates. See Box11-2 re. Cystic fibrosis (CF) and the CFTR.

I. Ion gradients provide the energy for 2E activetransport. See Table 11-4 & worked Example11-3.

J. Aquaporins form hydrophilic transmembranechannels for the passage of H2O.This is important.

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K. Ion-selective channels allow rapid movement ofions across membranes.

L. Ion-channel movement is measured electrically.

M. The structure of the K+ channel reveals thebasis for its specificity.

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N. Ion gated channels.

1. Neuronal Na+ channel is a voltage-gated ion channel.

2. The acetylcholine receptor is a ligand-gated ionchannel. Read on your own.

O. Defective ion channels can have adversephysiological consequences.

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