Chapter 9: Membranes and Membrane Transport

Passive diffusion is slow because…

ions and polar molecules do not readily cross the nonpolar part of the lipid bilayer

Passive diffusion must be…

facilitated by protein transporters

Lateral asymmetry

protein/lipid clusters in plane of membrane

Tranverse asymmetry

Difference in inner versus outer leaflet of membrane

Protein asymmetry is essential for function because:

1. Transporters must orient correct way

2. Receptor binding sites must orient to outside

3. Glycoproteins AND glycolipids (cell-cell recognition, immune response)

must face outside

Lipids are distributed asymmetrically because it is…

Important for correct protein-lipid interactions in asymmetrically-oriented protein

Movement of phosphatidylserine (PS) to the outer leaflet signals

Apopotosis

Aging erythrocytes move PS to outer leaflet leading to them being

engulfed by macrophages

Lateral lipid motion is

fast (exchange places at a rate of 10⁷/s)

Transverse or flip-flop motion is

Slower than lateral lipid motion (1/s or 1/day)

Flippase

Outer to inner leaflet (PS, in particular)

Floppase

Inner to outer leaflet

Flippase and Floppase

both are ATP dependent and are important to generate/maintain membrane asymmetry

Scramblase

Bidirectional, Ca²⁺-dependent

Destroy membrane asymmetry

Membrane protein lateral motion

1. Slower (few micron/s to 10 nm/s)

2. Association with other proteins or with cytoskeleton slows protein motion

3. Proteins do not flip-flop

Solid ordered or gel state, S_o (Low temperatures)

1. Alkyl chains fully extended (all anti)

2. Minimum surface area/lipid (head groups tightly packed)

3. Maximum bilayer thickness

Liquid crystalline state, L_d (High temperatures)

1. Alkyl chains more disordered (gauche conformers, causes kinks in alkyl chains)

2. Surface area/lipid increases

3. Bilayer thickness decreases (kinks = chains less extended)

S_o to L_d transition

1. True phase transition with a well-defined enthalpy

2. Sharp, i.e. all-or-none

3. Always endothermic

4. Characteristic melting temp. T_m

T_m increases

with chain length (more van der Waals)

T_m decreases

with unsaturation (poorer van der Waals)

Pre-transitions in membranes are observed due to

tilting of hydrocarbon chains

E. coli adjust lipid composition in response

to temperature change

How does E. coli respond to temperature change

sat./unsat. ratio from 1.6 to 1.0 causes temperature drops from 42 ^oC to 25 ^oC

Prevents membrane becoming too rigid

Natural membranes with a mix of lipids have

broader transitions

Cholesterol broadens transition/keeps T_m the same, which…

Allows eukaryotic membranes to function over broader temp range without changing lipid composition

L_o (liquid-ordered) phase observed in presence of cholesterol

-rigid cholesterol fused ring system sterically enforces order on nearby alkyl chains

-but poorer van der Waals so, still get good lateral motion

Regions of L_o phase occur in natural membranes are lipid rafts

Aggregates of cholesterol/ceramides and gangliosides/GPI-anchored proteins

In natural membranes lipids/proteins undergo

hop diffusion

Hop diffusion for lipids

15 ms

Hop diffusion for proteins

50 ms

Cytoskeleton (actin/spectrin filaments)

create compartments on inner membrane

(Fast diffusion on surface within compartment)

Membranes can be highly curved, for example

Cristae of inner mitochondrial membrane

Lipid composition can cause curvature through

-difference in number of lipids outer vs inner leaflet

-lipid shape (acyl chain/head group variation)

Proteins can cause curvature through

-conically shaped integral membrane proteins

-interaction with cytoskeleton proteins

-scaffolding proteins

BAR domains – dimeric banana-shaped proteins

• Scaffolding proteins

• Induce curvature when bound to plasma membrane

N-BAR

Insert N-terminal amphipathic helix causing curvature

Passive diffusion is slow because

ions and polar molecules do not readily cross the nonpolar part of the lipid bilayer

Properties of Facilitated Diffusion

1. Transport only in thermodynamically favorable direction

2. Specificity for a particular ion or molecule

K⁺ channels

Regulate cell volume, nerve impulses, hormone secretion

Typically very selective for K⁺ over Na⁺

KcsA, K⁺ channel from Streptococcus lividans

• Has 4 identical subunits each has two TM helices (inner and outer) + 3rd short helix (C- term – negative end helix dipole - ends at selectivity filter)

• Water-filled pore on extracellular side ends at selectivity filter

KcsA selectivity filter

• Pentapeptide (TVGYG) on interhelical loop from each subunit

• Thr side chain –OH and backbone carbonyls first 4 residues

KcsA Binding Site

• 4 K+ binding sites – each provide 8 O-atoms to coordinate to K+

• Replaces hydration sphere of K+ in H2O

• distance to O-atoms fits ionic radius K+

K⁺ ionic radius

1.33 Å

Na⁺ ionic radius

0.95 Å

High conduction despite high selectivity in KcsA is due to

1. Repulsion closely spaced K⁺

2. At high K⁺ conformational change = more symmetrical pore

KcsA is channel gated by pH

• Closed at neutral pH opens at low pH

• Long inner helix bends at a conserved glycine opens pore on intracellular side

Some channels have pore within each subunit

Cl- (dimer), NH3 (trimer), glycerol, (tetramer)

NH3 channel is hydrophobic

lowers pKa of NH4+ to allow transport as NH3

Glycerol channel is

amphipathic interact methylenes of glycerol one side, -OH’s on other

primary active transport

generates ion or H+ gradients using energy derived from ATP hydrolysis or light

secondary active transport

using ion or H+ gradient to drive transport of nutrients/waste

electrogenic active transport

leads to a net transport of charge across a membrane

P-type ATPase common features

-E1 form open to one side

-E2-P form open to other

-E2-P has phosphorylation at a specific Asp by ATP that drives a

conformational change (converts chemical energy of ATP to

mechanical energy of conformational change)

Na+,K+-ATPase

keeps Na+ low and K+ high in cytoplasm (gradients used in excitation

of neurons/muscle cells, control of cell volume, transport sugars and a.a.’s)

Ca²⁺-ATPase

keeps [Ca²⁺] at 0.1 microM in cytoplasm, 1.5 mM in sarcoplasmic reticulum

(SR) of muscle cells. Gradient used in muscle contraction

Gastric H+,K+-ATPase

keeps pH gradient across mucosal membrane of 6.6 pH units

Ca²⁺-ATPase cytoplasmic domains

• N, binds ATP

• P, site of Asp 351 that phosphorylated by ATP

• A, actuator domain, transmits conformational changes in cytoplasmic domains to M domain

Ca²⁺-ATPase Transmembrane Domain

M domain – 10 TM α-helices, two Ca2+ sites provide 7 O-atoms to each (Asp, Asn, Glu, Thr, H2O, main chain C=O)

Ca²⁺-ATPase Catalytic cycle:

1. Ca2+ binds from cytoplasmic side

2. ATP binds, N and A domains close around P domain – M domain unaffected

3. ATP transfers phosphate to Asp351

4. ADP dissociates: E2-P conformer now stabilized, Ca2+ binding sites disrupted transporter open to SR – 2 Ca2+ enter SR

5. Asp-CO-O-PO3 hydrolyzed, Pi released

6. E2 returns to E1 conformer

Bacteriorhodopsin, bR, from Halobacterium halobium

• 7 TM helices

• Color from retinal cofactor attached as a Schiff’s base to Lys216 middle of membrane

• when shine light on retinal: trans to cis isomerization

bR conformational change caused in protein favors

Transfer H+ from protonated Shiff base to Asp85 (to extracellular medium)

cis-retinal reverts to trans oriented to

pull H+ off Asp 96 (Asp96 then reprotonated from cytoplasm)

symports

-H+/Na+ transported in same direction as transported nutrient

-typically for sugar and a.a transport

antiports

-H+/Na+ move in opposite direction of species transported

-Na+-Ca2+ antiporter supplements Ca2+-ATPase

Cytosine

Uracil

Thymine

Adenine

Guanine

Uracil keto-enol tautomerization

For uracil and thymine the ____ is favored at neutral pH

keto form

pKa’s of N-1 and N-3 > ___ for uracil

8

pKa(N-3) = ___ for uracil

9.5

Cytosine enol form (-NH2 at C4, not an imine C=NH) predominates

neutral pH (pKa(N-3) = 4.5, deprotonated at neutral pH)

keto-enol tautomerization for guanine

For guanine ___ tautomer predominates

keto

pKa(N-1) = 9.4

pKa(N-3) < 5

pyrimidine

purine