Transport across membranes

Categories of Transport An important function of membranes is to selectively

control movement of substances in and out of compartments

For transport of organic metabolites and inorganic ions, three transport processes are utilized Cellular transport: Transport of materials (raw

material, waste ansd secretory products) across plasma membrane

Intracellular transport: Transport of substances across membranes of organelles eg nucleus, mitochondrion etc

Transcellular transport: uptake of substances from one cell to the other eg movement of glucose through the epithelial cells from the small intestine to the blood

Membrane Semi- Permeability

The Plasma Membrane is semi-permeable ( also referred as “selectively permeable” ).

A selectively permeable membrane allows the passage of some materials in or out of a cell, and restricts the passage of some

materials in or out of the cell.

Semi - Impermeable Membranes

Permeability of plasma membraneGeneral principles II

Permeability coefficient (cm/sec)

Mechanisms of membrane transport

Membrane transport can either be: Passive diffusion: do not require energy.

Molecules move from higher to lower concentration

Facillitated transport; Similar to passive transport but differ in that carrier proteins are required

Active transport

Passive Transport Simple diffusion

diffusion of nonpolar, hydrophobic molecules lipids high low concentration gradient

Facilitated transport diffusion of polar, hydrophilic molecules through a protein channel

high low concentration gradient Active transport

diffusion against concentration gradient low high

uses a protein pump requires ATP

Getting through cell membrane

Getting through cell membrane

Three Forms of Transport Across the MembraneThree Forms of Transport Across the Membrane

Passive transport Passive transport refers to movement of a

solute through a membrane dictated by the existing gradient or charge ie down the concentration gradient

The free energy change ΔG is always negative (exergonic) ie ΔG<0, negative free energy change

Passive transport is inherently non-directional with respect to the membrane ie the solute can move inward or outward depending on the prevailing concentration or electrochemical gradient

Simple Diffusion

Diffusion is the flow of substances from an area, or region, of greater molecular concentration

to an area, or region, of lesser molecular concentration. The overall direction of the movement is referred to as the Gradient.

Molecules usually move “down the concentration gradient”..... flow from high

concentration to low concentration. Eventually a state of “equilibrium” is reached where molecules are uniformly distributed but

continue to move randomly.

Simple DiffusionSimple Diffusion is a passivepassive process ( nono energy required).

Some substances will diffuse through membranes as if the membranes weren’t even there.

Molecules diffuse until they are evenlyevenly distributed.

The molecules move from an area of [high] to [low].[high] to [low].

EXAMPLES of molecules that easily cross cell membranes by simple diffusion are: oxygen, carbon dioxide, alcohols, oxygen, carbon dioxide, alcohols, fatty acids, glycerol, and ureafatty acids, glycerol, and urea.

Simple diffusion

Alcohol & Urea

Fatty Acids, Glycerol,

Simple diffusion

• Factors governing simple diffusion and membrane permeability– Size : Lipid bilayer is more permeable to smaller

molecules eg H20 more permeale than urea. Small molecules such as H20, C02 and 02 diffuse freely while large molecules eg glucose require carriers

– Polarity : Bilayer more permeable to non-polar solutes and less permeable to polar solutes ie non polar molecules more readily pass in the non-polar phase of lipid bilayer eg propanol is more permeable than gycerol

– Ions : Bilayer highly impermeable to ions eg 02 more permeable than hydroxyl ion 0H-. Energy is required to move ions from aqueous to non-polar environment

Simple Diffusion• What are the major factors that influence

diffusion across a membrane?

The rate of diffusion will be increased when there is :

1.1. ConcentrationConcentration: the difference in [ ] between two areas (the [ ] gradient) causes diffusion. The greater the greater the difference in concentration,difference in concentration, the fasterfaster the diffusion.

2.2. Molecular sizeMolecular size: smaller substances diffuse more quickly.quickly. Large molecules (such as starches and proteins) simply cannot diffuse through.

3. Shape of Ion/MoleculeShape of Ion/Molecule: a substance’s shape may preventprevent it from diffusing rapidly, where others may have a shape that aids their diffusion.

Simple diffusion

4. 4. Viscosity of the MediumViscosity of the Medium: the lower the viscosity, the more slowlyslowly molecules can move through it.

5.5. Movement of the MediumMovement of the Medium: currents will aid diffusion. Like the wind in air, cytoplasmic steamingcytoplasmic steaming (constant movement of the cytoplasm) will aid diffusion in the cell.

6.6. SolubilitySolubility:: lipidlipid - soluble molecules will dissolve through the phospholipid bilayer easily,easily, as will gases like CO2 and O2.

7. PolarityPolarity: waterwater will diffuse, but because of its polarity, it will

notnot pass through the non-polar phospholipids. Instead, water passes though specialized protein ionprotein ion channels.

Simple diffusion

Simple diffusion : Summary

Apply generally to small molecules (uncharged) Does not require carrier – only require a carrier if

molecule transported is charged Move substances in the direction dictated by the

concentration gradient for the substance Characterized by a linear relationship between

the concentration gradient and the rate of movement across the membrane, with no evidence of saturation at higher concentrations

Diffusion Through the Plasma Membrane

Carrier Mediated Transport

Many molecules cannot enter or leave cell by diffusion

CMT utilizes proteins to carry solutes across cell membrane

Characteristics of mediated transport:1. Specificity - each transport protein binds to and

transports only a single type of molecule or ion2. Competition - results from similar molecules binding to

the same protein.3. Saturation - rate of movement of molecules is limited

by the number of available transport proteins

Membrane Carriers

Uniporter carries only one solute at a time

Symport carries 2 or more solutes simultaneously in

same direction (cotransport) Antiport

carries 2 or more solutes in opposite directions (countertransport)

sodium-potassium pump brings in K+ and removes Na+ from cell

Any carrier type can use either facilitated diffusion or active transport

Carrier proteinsProteins that act as carriers are too large to move across the membrane.

They are transmembrane proteins, with fixed topology.

An example is the GLUT1 glucose carrier, in plasma membranes of various cells, including erythrocytes.

GLUT1 is a large integral protein, predicted via hydropathy plots to include 12 transmembrane -helices.

Carrier proteins cycle between conformations in which a solute binding site is accessible on one side of the membrane or the other.

There may be an intermediate conformation in which a bound substrate is inaccessible to either aqueous phase.

With carrier proteins, there is never an open channel all the way through the membrane.

conformation

change conformation

change

Carrier-mediated solute transport

Carrier proteins

The transport rate mediated by carriers is faster than in the absence of a catalyst, but slower than with channels.

A carrier transports one or few solute molecules per conformational cycle, whereas a single channel opening event may allow flux of many thousands of ions.

Carriers exhibit Michaelis-Menten kinetics.

conformation

change conformation

change

Carrier-mediated solute transport

Carrier proteins

Saturation of a Carrier Protein

1. When the concentration of x molecules outside the cell is low, the transport rate is low because it is limited by the number of molecules available to be transported.

2. When more molecules are present outside the cell, as long as enough carrier proteins are available, more molecules can be transported; thus, the transport rate increases.

3. The transport rate is limited by the number of carrier proteins and the rate at which each carrier protein can transport solutes. When the number of molecules outside the cell is so large that the carrier proteins are all occupied, the system is saturated and the transport rate cannot increase.

Classes of carrier proteins

Uniport (facilitated diffusion) carriers mediate transport of a single solute.

An example is the GLUT1 glucose carrier.

The ionophore valinomycin is also a uniport carrier.

Uniport Symport Antiport

A A B A

B

A gradient of one substrate, usually an ion, may drive uphill (against the gradient) transport of a co-substrate.

It is sometimes referred to as secondary active transport.

E.g: glucose-Na+ symport, in plasma membranes of some epithelial cells bacterial lactose permease, a H+ symport carrier.

Symport (cotransport) carriers bind two dissimilar solutes (substrates) & transport them together across a membrane.

Transport of the two solutes is obligatorily coupled.

A substrate binds & is transported.

Then another substrate binds & is transported in the other direction.

Only exchange is catalyzed, not net transport.

The carrier protein cannot undergo the conformational transition in the absence of bound substrate.

Antiport (exchange diffusion) carriers exchange one solute for another across a membrane.

Usually antiporters exhibit "ping pong" kinetics.

Example of an antiport carrier:

Adenine nucleotide translocase (ADP/ATP exchanger) catalyzes 1:1 exchange of ADP for ATP across the inner mitochondrial membrane.

ATP 4

ADP 3

mitochondrial matrix

adenine nucleotide translocase

Carrier Mediated Transport: Facilitated Diffusion

• Glucose and amino acids are insoluble in lipids and too large to fit through membrane channels

• Passive process, i.e. no ATP used• Solute binds to receptor on carrier protein

– Latter changes shape then releases solute on other side of membrane

– Substance moved down its concentration gradient

Facilitated diffusion

• Facilitated transport differs from simple diffusion in that carrier proteins are required

• Carrier proteins are Integral membrane proteins (specific for a single compound or a small group of closely related compounds

• Carrier proteins sometimes referred to as permeases or transport proteins

Permeases

• Permeases become saturated as concentration of transported solute is raised

• Permeases catalyse the facilitated diffusion of only one or a few structurally related solutes

• Like enzymes , they are subject to competitive inhibition by molecules or ions that are structurally related to the substrate being transported

Facilitated diffusion

• Require 3 stages– Binding of solute or substrate– Physical translocation of solute (substrate).

Polar groups of solute shielded from non-polar interior of the membrane

– Release of product

Facilitated TransportFacilitated Transport: Some molecules are notnot normally able to pass through the lipid membrane, and need channel or carrierchannel or carrier proteinsproteins to help them move across.

This does notnot require energyrequire energy when moving from [H] to [L] (with the concentration gradient).

Molecules that need help to move through the plasma membrane are either charged, polar, charged, polar, or too large or too large..

Facilitated diffusion

If molecules are POLAR, CHARGED, or TOO LARGE they need a protein to help them across the membrane

EXAMPLES: sugars, amino acids, ions, nucleotidessugars, amino acids, ions, nucleotides ….

Each protein channel or protein carrier will allow only ONE TYPE ONE TYPE OF MOLECULEOF MOLECULE to pass through it.

Specificity of carriers and channels

Many channels contain a “gate”“gate” which control the channel's permeability.

When the gate is open, the channel transports, and when the gate is closed, the channel is closed.

These gates are extremely important in the nervenerve cells.

Gated channels

Diffusion and facilitated diffusion

Facilitated diffusion-rate reaches maximum when carrier is saturated

Pores saturated

Rate of transport

Concentration

Simple diffusion- rate Proporttional to [solute]

Carrier Mediated Transport: Active Transport

Uses ATP to move solutes across a membrane It is not dependent on a [ ] gradient

Can move substances against their [ ] gradients - i.e. from lower to higher concentrations!

Allows for greater accumulation of a substance on one side of the membrane than on the other.

Carrier proteins utilized called ion or exchange pumps. Ion pumps: actively transport Na+, K+, Ca++, Cl-

Exchange pumps: Na+-K+ pump

Active transportActive transport

Is the movement of solute against or up a concentration gradient. i.e from a compartment of

low concentration to a compartment of high concentration.

Entropy will decrease (the solute become less random) and the free energy of the system will increase. ΔG>0, +ve free energy change (ΔG+,

endergonic) Active transport is a process in which the system

gains free energy. Active transport is unidirectional ie movement is in

one direction only. Transport of the same solute in the other direction is impossible

Active TransportActive Transport: the movement of polar, large, and charged molecules moving againstagainst the [ ] gradient (uphilluphill).

EXAMPLES of molecules that move this way are all of the things that require protein carriers to move across the plasma membrane.

ionsions (like Na+ and K+ in cells, and iodine) and sugarssugars, amino acidsamino acids, nucleotidesnucleotides...

Active transport

Active transport

The active transport pumps must provide translocation of the solute and couple this translocation to an energy yeilding reaction ie hydrolysis of ATP

However, this is not always the case because the driving force for active transport in some cases may be an ion gradient

Energy source of active transport

Supplied as a high energy phosphate bond in form of ATP

Other compounds also used eg sugar transport in bacterial cells depending on PEP as an energy source

Cotransport-depending on the electrochemical gradient of either Na+ or H+ to drive active symport of a give solute

Active transport Serves three major functions

Uptake of fuel molecules and essential nutrients from the environment even when in low concentrations

Removal of secretory products and ions from cell organelles even when concentrations outside are higher

Enables the cell to maintain constant and optimal internal concentrations of ions such as H+, K+, Na+ Ca2+ etc

Active Transport

Active transport

-requires energy – ATP is used directly or indirectly to fuel active transport

-moves substances from low to high concentration

-requires the use of carrier proteins

Active Transport

Carrier proteins used in active transport are the same used in facilitated transport and include:-uniporters – move one molecule at a time-symporters – move two molecules in the same direction-antiporters – move two molecules in opposite directions

Carrier proteins

This classification is independent of whether the transport is active or passive.

Active Transport• Energy coupling can transport against a

concentration gradient

PrimaryTransport is coupled to a chemical process (ATP hydrolysis)- Utilizes the energy of ATP hydrolysis to transport a molecule against its concentration gradient

SecondaryTransport is coupled to a favorable transport process- Utilizes the downhill flow of one gradient to power the formation of another gradient

ATP Powered pumps• Four classes of ATP powered pumps

– P-class pump : found in plasma membranes of eukaryotes (Na+/K+ pump; Ca2+ pump); Mammalian stomach (H+/K+ pump); Muscle cells (Ca2+ pump). Catalytic domain become phosphorylated as part of transport cycle

– V-class pumps : found in vacuolar membranes of eukaryotes and endosomal and lysosomal membranes of animal cells as well as osteoclasts and kidney tubule cells. V-class pumps couple ATP hydrolysis to transport protons against a concentration gradient.

– F class pumps: found in bacterial membrane, inner mitochondrial membrane and thylakoid membrane of chloroplasts. F-class pumps utilize energy in a proton concentration or eletrochemical gradient to synthesize ATP. Note – V and F class pumps do not form phosphoprotein complexes like the P- class. Their structures are similar with similar subunits which are unrelated with the P-class pumps

– ABC (ATP binding cassette) superfamily : Found in bacterial plasma membrane (transports sugars, amino acids and peptides); mammalian plasma membrane (transports phospholipids, drugs , cholesterol, small molecules). ABC proteins contain 2 transmembrane domains and 2 cytosolic ATP binding domains that couple ATP hydrolysis to solute transport

ATP powered Pumps

Use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient or electric potential.

Overall reaction—ATP hydrolysis and the “uphill” movement of ions or small molecules—is energetically favorable

P, F, and V classes transport ions only, whereas the ABC superfamily class transports small molecules as well as ions.

F-type ATPases - Proton Gradients <==> ATP

• Can either use ATP to pump protons or proton gradients to make ATP

ABC transporters - homologous family• classified by sequence and structure - not by function• ATP dependent transport

– Multidrug resistance transporter pumps out foreign compounds

– The chloride channel CFTR responsible for cystic fibrosis

– Flippases for transbilayer lipid transport

Active Transport The four types of transport ATPases

Active transport driven by an ion gradients

Ion Gradients - Na+ or H+ can drive secondary transport

• lac permease - bacterial lactose proton symport– Active transport of Lactose depends on maintenance

of proton gradient

Secondary Active

Transport

• Ions or molecules move in same (symport) or different (antiport) direction.

• Is the movement of glucose a symporter example or an antiporter example?

• This example shows cotransport of Na+ and glucose. 1. A sodium-potassium

exchange pump maintains a concentration of Na that is higher outside the cell than inside. Active transport.

2. Na moves back into the cell by a carrier protein that also moves glucose. The concentration gradient for Na provides the energy required to move glucose against its concentration gradient.

Na+- Glucose Symport in human intestine

• 2 Na+out + Glucoseout --> 2

Na+in + Glucosein

• Combination of sodium chemical potential and membrane potential

• provide driving force for ~9000 fold concentration [Glucose]in/[Glucose]out

Binding of cytoplasmic Na+ to the pump protein stimulates phosphorylation by ATP.

1

2

3

4

Phosphorylation causes the protein to change its shape.

The shape change expels Na+ to the outside, and extracellular K+ binds.

5

Loss of phosphate restores the original conformation of the pump protein.

K+ binding triggers release of the phosphate group.

6K+ is released and Na+ sites are ready to bind Na+ again; the cycle repeats.

Concentration gradients of K+ and Na+

Extracellular fluid

Cytoplasm

Sodium-Potassium Pump

Functions of Na+ -K+ Pump• Regulation of cell volume

– “fixed anions” attract cations causing osmosis– cell swelling stimulates the Na+- K+ pump to

ion concentration, osmolarity and cell swelling

• Heat production (thyroid hormone increase # of pumps; heat a by-product)

• Maintenance of a membrane potential in all cells– pump keeps inside negative, outside positive

• Secondary active transport (No ATP used)– steep concentration gradient of Na+ and K+ maintained

across the cell membrane– carriers move Na+ with 2nd solute easily into cell

• saves glucose in kidney

Ionophores and membrane transport

Ionophores are compounds, usually antibiotics that greatly increase the permeability of membranes to specific ions

They function by two processes: Providing a hydrophilic channel through the

hydrophobic membrane bilayer through which the ion pass through (channel formers eg Gramicidin)

Surrounding the ion to be transported with a hydrophobic coat making it lipid soluble (ion carrier eg Valinomycin)

Gramicidin A polypeptide of about 15 aa that inhibit growth of

gram positive bacteria Protein consists of alternating L and D amino acids When gramicidin inserts into membranes it

assumes a helical shape Hydrophobic groups are in contact with

membrane phospholipids, polar groups on the inside forming a hydrophilic lining

Two gramicidin monomers associate end to end forming a 0.4nm continuous channel through which K+ ions pass through at a late of 10^6 ions per second

Channels cycle between open & closed conformations.

When open, a channel provides a continuous pathway through the bilayer, allowing flux of many ions.

Gramicidin is an example of a channel.

closed

conformationchange

open

Ion Channels

Gramicidin is an unusual peptide, with alternating D & L amino acids.

In lipid bilayer membranes, gramicidin dimerizes & folds as a right-handed -helix.

The dimer just spans the bilayer.

Primary structure of gramicidin:

Gramicidin dimer(PDB file 1MAG)

HCO-L-Val-D-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-NHCH2CH2OH Note: The amino acids are all hydrophobic; both peptide ends are modified (blocked).

The outer surface of the gramicidin dimer, which interacts with the core of the lipid bilayer, is hydrophobic.

Ions pass through the more polar lumen of the helix.

Ion flow through individual gramicidin channels can be observed if a small number of gramicidin molecules is present in a lipid bilayer separating 2 compartments containing salt solutions.

Gramicidin dimer(PDB file 1MAG)

An open channel forms when two gramicidin molecules join end to end to span the membrane.

This model is consistent with the finding that at high [gramicidin] overall transport rate depends on [gramicidin]2.

Gating (opening & closing) of a gramicidin channel is thought to involve reversible dimerization.

Valinomycin Valinomycin : a mobile ion carrier ionophore

Ring shaped polymer protein with hydrophobic exterior (made of valine side chains). These interact with hydrophobic core of lipid bilayer

Contain six oxygen atoms attached to carbonyl atoms of valines. The oxygen atoms bind the ion, stabilizing it within the ring cavity as the ionophore diffuses across the memebrane ie valinomycin provides a hydrophilic environment for an ion as it traverses the memebrane - moving across the membrane with the ion

Valinomycin is a carrier for K+.

It is a circular molecule, made up of 3 repeats of the sequence shown above.

N C H C OHC

C H

C H 3H 3 C

O

C N

C H

C H 3H 3 C

OHC

C H

C H 3H 3 C

C O C H

C H 3

C

O

H

O

H

3

V a lin o m y c in

L -v a l in e D -h y d ro x y - D -v a l in e L - la c t ic i s o v a le r ic a c id a c id

Valinomycin is highly selective for K+ relative to Na+.

The smaller Na+ ion cannot simultaneously interact with all 6 oxygen atoms within valinomycin.

Thus it is energetically less favorable for Na+ to shed its waters of hydration to form a complex with valinomycin.

Valinomycin

O O O

O O

Hydrophobic

O

K+

Puckering of the ring, stabilized by H-bonds, allows valinomycin to closely surround a single unhydrated K+ ion.

Six oxygen atoms of the ionophore interact with the bound K+, replacing O atoms of waters of hydration.

Whereas the interior of the valinomycin-K+ complex is polar, the surface of the complex is hydrophobic.

This allows valinomycin to enter the lipid core of the bilayer, to solubilize K+ within this hydrophobic milieu.

Valinomycin

O O O

O O

Hydrophobic

O

K+

Valinomycin is a passive carrier for K+. It can bind or release K+ when it encounters the membrane surface.

Valinomycin can catalyze net K+ transport because it can translocate either in the complexed or uncomplexed state.

The direction of net flux depends on the electrochemical K+ gradient.

Val Val

Val-K+ Val-K+

K+

membrane

K+

Summary of Transport Summary of Transport TypesTypes