TransportThrough Membranes

Necessity for Transport

• Plasma Membranes

• Intracellular Membranes (Organelles)

Biological Membranes

• Organized assemblies of lipids, proteins and small amounts of carbohydrates

• Regulate composition of intracellular medium by controlling flow of nutrients, waste products, ions, etc. in and out of cell

• Scaffolding– Oxidative phosphorylation– Photosynthesis– Nerve impulses– Hormone receptors

Membrane Lipids

Generally soluble in organic solvents

Types of Membrane Lipids

• Glycerophospholipids

• Sphingolipids• Cholesterol

Glycerophospholipids(Glycerol 3-phosphate)

H2C

CH

H2C OPO32-

OH

HOGlycerol

Phosphate

Phosphatidic Acid(1,2-diacylglycerol-3-P)

O

CH

H2C OPO32-

C R1H2C

O

OCR2

OFatty Acids

Membrane Glycerophospholipids

O

CH

H2C O

C R1H2C

O

P

O

O–

O R3 Alcohol

Fatty Acids

OCR2

O

Glycerol

Fatty Acids

• Saturated and unsaturated

• Amphiphilic

COO–

Glycerophospholipid Alcohols

• Ethanolamine

• Serine

• Choline

H O C H2

C H2

N H3

+

H O C H2

C H

N H3

+

C O O

–

H O C H2

C H2

N ( C H3

)3

+

Amphiphilicity

Nonpolar Tail

(Hydrophobic)

Polar Head

(Hydrophilic)

Alcohol

P

Glycerol

Sphingolipids(Sphingosine)

CH3(CH2)12 CH CH CH

OH

CH

CH2OH

NH2

Sphingolipids(Sphingomyelin)

C H3

( C H2

)1 2

C H C H C H

O H

C H

C H2

N H C R1

O

O P

O

O

–

O C H2

C H2

N ( C H3

)3

C h o l i n e

+

F a t t y A c i d

Myelin Sheath – defective in some metabolic diseases

Cholesterol

H O

C H3

C H3

C H

C H3

C H2

C H2

C H2

C H

C H3

C H3

F l e x i b l e H y d r o p h o b i c T a i lH y d r o p h i l i c

( P o l a r H e a d )

R i g i d F u s e d R i n g

Properties of Cholesterol

• Highly hydrophobic (low solubility)

• Lowers melting point of mixed lipids

• Stiffens surface proximal region of bilayer

Occurrence of Cholesterol

• Animal plasma membranes• Organelle membranes (some)• Precursor to steroid

hormones

Properties of Lipid Aggregates

Monolayers, Micelles and Bilayers

Monolayers(very dilute solutions)

Water

Polar Head Groups

Hydrophobic Tails

Air

Micelles(single-tailed lipids)

W e d g e - S h a p e d

( V a n d e r W a a l s e n v e l o p e )

Critical Micelle Concentration

Cylindrical Lipids

Nonpolar Tail

(Hydrophobic)

Polar Head

(Hydrophilic)

Alcohol

P

Glycerol

Bilayers

6 0 Å

O u t e r L e a f l e t

I n n e r L e a f l e t

H y d r o p h o b i c

T a i l s

H y d r o p h i l i c

H e a d s

A q u e o u s P h a s e

A q u e o u s P h a s e

Liposomes

P h o s p h o l i p i d B i l a y e r

Properties/Uses of Liposomes

Single Bilayer(inner and outer leaflets)

Delivery of Therapeutic Agents

Membrane Proteins

Peripheral or Extrinsic Proteins

Integral or Intrinsic Proteins

Peripheral or Extrinsic Proteins

• Easily dissociated– High ionic strength– pH chages

• Free of attached lipid (dissociated)

• Water-soluble (e.g. cytochrome c)

• Normal amino acid composition

Integral or Intrinsic Proteins

• Not easily dissociated• Retain associated lipid (dissociated)• >average hydrophobic amino acds• Significant number hydrophilic amino

acds• Asymmetrically oriented amphiphiles• Trans-membrane proteins

Asymmetric Orientation

Integral or Intrinsic Proteins

Membrane Carbohydrates

• Mostly oligosaccharides

• Variety of sugars• Often sialic acid• Glycolipids• Glycoproteins

Membrane Structure

Fluid Mosaic Model

Carbohydrate

Peripheral or

Extrinsic Protein

Integral

or

Intrinsic

Protein

Integral

or

Intrinsic

Protein

OUTSIDE

INSIDE

Lipid

Bilayer

Membrane Permeability(Impermeable to Most Polar Substrates)

Aqueous solutions

Water-soluble compound

Membrane

Thermodynamicsof

Transport

Move Toward Equilibrium

Move from Higher Concentration

toLower Concentration

No information about rate of movement!

Kinetics and Mechanisms

ofTransport

Types of Transport

• Nonmediated Transport (Diffusion)• Mediated Transport (Transport

Proteins)– Passive-mediated Transport

(facilitated diffusion)– Active Transport

Terminology

• Carriers

• Permeases

• Porters

• Translocases

• Translocators

• Transporters

Non-mediated Transport(Permeability Coefficient)

Rate

[A]out

– [A]in

Rate

[A]out

[A]out

> [A]in

[A]in

= Constant

Mediated Transport(Transport Proteins)

Rate

[A]

Mediated Transport Properties

• Saturation kinetics• Speed and specificity• Susceptibility to

competitive inhibition• Susceptibility to

chemical inactivation

Asymmetric Orientation

Types of Transporters

Ionophores

Valinomycin

Gramicidin

Stoichiometry

Electrical Character

• Electroneutral

• Electrogenic

Passive-MediatedGlucose Transport

Erythrocyte Glucose Transporter

Asymmetric Orientation

Conformational Change

Insulin Action(Muscle and Adipocytes)

ATP-Driven Active Transport

Against a concentration gradient

Often coupled to ATP hydrolysis

Na+-K+-ATPase of Plasma Membrane(Reaction)

3 Na+(in) + 2 K+(out) + ATP + H2O

3 Na+(out) + 2 K+(in) + ADP + Pi

Electrogenic Antiport

2 K+

3 Na+

Outside

Inside

Mechanism

2 K+

3 N a+

2 K+

3 N a+

3 N a+

3 N a+

3 N a+

2 K+ – P~ PA T P

M g2 +

M g2 +

A T PA D P3 N a

+

2 K+ H

2O

A T P

B i n d i n g

~ P

F o r m a t i o n

o f

A s p a r t y l ~ P

Pi

N a+

T r a n s p o r t

N a+

R e l e a s e

K+

B i n d i n g

P h o s p h a t e H y d r o l y s i s a n d K+

T r a n s p o r t

N a+

B i n d i n g

K+

R e l e a s e

I N S I D E

O U T S I D E

Mechanism

Mechanistic Steps

Na+-dependent ATP Phosphorylation of Aspartic Acid Side Chain

K+-dependent Hydrolysis

Mechanistic Evidence

C

HC

O

CH2

C

NH

O

OPO3

2+

Aspartyl Phosphate Residue

LiB H4

H

+ C

HC

O

CH2

C

NH

H

OH

H

Homoserine

Group Translocation

Simultaneous Transportand

Chemical Modification

PEP-dependent Phosphotransferase System

(PTS)

PEP

Pyruvate

EI

EI~P HPr

HPr~P EIIIg

EIIIg

~P

Glucose

Glucose-6-P

ATP

PPi

+ cAMP

Adenylate

Cyclase

Activation

EIIg

COOH

C

CH2

OP

Soluble

Cytoplasmic

Proteins

(Common)

EIIIg

Inhibits Lactose Permease

Inhibits Glycerol Kinase

Hpr: Histidine-containing phosphocarrier protein

C

HC

O

CH2

NH

N

N

P O

–

O

O

–

Phosphohistidine

Ion Gradient-DrivenActive Transport

Free Energy of Electrochemical Gradient

(Established by ion-pumping)Power Endergonic Physiological

Processes

Na+-Glucose Symport(Mechanism)

N a

+

+ G l u c o s e

N a

+

+ G l u c o s e

O u t s i d e

I n s i d e

N a

+

G l u c o s e

N a

+

G l u c o s e

C o n f o r m a t i o n a l C h a n g e

a n d

T r a n s p o r t R e l e a s e

C o n f o r m a t i o n a l C h a n g e s

Na+-Glucose Symport(Function)

Na

+

Na

+

K

+

K

+

Glucose

Glucose Glucose

Intestinal Lumen Capillaries

Brush

Border

Cell

Na

+

–glucose symport

Na

+

–K

+

–ATPase

Glucose uniport

Lactose Permease

H

+

+ L a c t o s e

H

+

+ L a c t o s e

O u t s i d e

I n s i d e

H

+

L a c t o s e

H

+

L a c t o s e

B i n d i n gT r a n s p o r t

R e l e a s e

R e c o v e r y

C o n f o r m a t i o n a l C h a n g e s