AH Biology: Unit 1

Cells and Proteins

Membrane Proteins:

Channel and Transport Proteins

Transport proteinsTransport proteins can be characterised by the nature of the role they play.

Uniports simply transport a molecule from one side of the membrane to another down concentration or electrochemical gradients

Active transport•Symports are examples of coupled transport where the transporting of one solute results in the coupled transport of another in the same direction, eg glucose/Na+ symport.

•Antiports are an example of coupled transport of two molecules being transported in opposite directions, eg Na+/K+ ATPase.

Uniport Symport Antiport

Transporter proteinsTransporter proteins change shape (conformation) in order to transport molecules across membranes.

This may be passive (simply facilitate diffusion down a gradient) or active (transport against the gradient).

This energy can come from:

•ATP, eg Na+/K+ ATPase

•electrochemical gradients, eg glucose/Na+ symport

•light-driven pumps, eg bacterial rhodopsin.

Passive transporters

• The transporter exists in two reversible states which cycle from A to B randomly.

• Cycling is not dependant on solute binding.

• If the solute concentration is higher on one side than the other molecules are transported in line with their gradient.

• GLUT 1, 3 and 4 proteins are located on the plasma membranes of cells and are good examples of passive glucose transporters.

• They have high-affinity binding sites for glucose and readily facilitate uptake of glucose down its concentration gradient.

Na+/K+ ATPase

• This transporter is mainly responsible for maintaining an Na+/K+ gradient across the plasma membrane.

Na+/K+ ATPase

• The transporter has binding sites with high affinity for three Na+ ions.

• When Na+ binds, ATP is reduced, transferring a phosphate to the transporter. The resulting enforced conformational change opens the transporter to the extracellular side, pumping the Na+ ions across the membrane as conformational change reduces affinity for

• Two binding sites with affinity for K+ are then exposed to the extracellular side.

Na+/K+ ATPase

• Two K+ ions bind from the extracellular side.

• This results in the transporter being dephosphorylated and the phosphate being released.

• The transporter returns to its initial state.• The K+ is pumped into the cytoplasm.• The affinity for Na+ on the inside of the

membrane is restored

Na+/K+ ATPase

• As both sodium and potassium are pumped against their concentration gradient this system is an example of active transport driven by ATP hydrolysis.

• As three Na+ are expelled for every two K+ this contributes to the membrane potential of the cell, which has a higher net negative charge intracellularly.

• Cells contain a large concentration of solutes plus a large number of negatively charged molecules (eg DNA) which are balanced by other cations within the cell. This creates a huge osmotic pull, driving water inwards by osmosis through the membrane and via water channels, eg aquaporins.

• Animal cells can counteract this osmotic pull to some degree by maintaining high extracellular concentrations of ions such as Na+ and Cl–. The Na+/K+ ATPase helps maintain osmolarity/tonicity by pumping out any sodium that leaks in down its high concentration gradient. Negatively charged chloride is excluded by the membrane potential.

• The maintenance of ion gradients by this transporter is responsible for around 25% of the basal metabolic rate of cells.

Glucose/Na+ symport

Glucose/Na+ symportThere are two classes of binding sites, one for Na+ and the other for glucose. Binding of either molecule enhances the binding of the other. As this system is driven by the Na+ gradient generated by the Na+/K+ ATPase it is described as secondary active transport.

When all binding sites are filled a conformational change in the protein delivers both molecules across the membrane. Later the sodium is pumped back out of the cell by the Na+/K+ ATPase. Because the conformational change relies on both sets of sites being filled or not the switch between states only happens if all sites are full or empty. This transport protein exists in two states A and B.

Because of the much higher extracellular than intracellular Na+ levels, glucose is more likely to bind to the molecule in the A state than the B state. More glucose and Na+ enter the cell by A–B transitions than are lost by the reverse. This is an example of cooperative co-transport.

This net flow results in an accumulation of glucose against its concentration gradient. The sodium ions flow down their electrochemical gradient while the glucose molecules are pumped up their concentration gradient.

The Na+/glucose symport is used to actively transport glucose out of the intestine and also out of the kidney tubules and back into the blood.

Channel proteins

Channel proteins exist as passive facilitators of molecule movement. Examples are:

• aquaporins (water channels)

• ligand-gated ion channels

• voltage-gated ion channels.

Aquaporins

Aquaporins are found in many disparate organisms, eg animals, plants and bacteria.

• The basic protein structure consists of tetramers each with a pore at its centre. Hydrophilic amino acids line one side of the pore while hydrophobic amino acids form the outside of the molecule embedded in the lipid bilayer.

• Aquaporins are not generally gated channels, but some plant species can exert control over aquaporins in response to dehydration. This results in closure of the channels.

• Each subunit, eg in aquaporin 1, consists of six membrane-spanning domains primarily consisting of alpha helices. Folding of the protein creates a pore through which water can pass, although it does not transport ions. This selection is brought about by producing a pore with hydrophobic amino acids on one side and hydrophobic opposite. This narrowing of the pore results in the inability of any hydrated ion to enter as it would be too large.

Aquaporins

Structure of AQPZhttp://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0000072#s3

Aquaporin Z (AQPZ) showing classic tetramer structure and alpha-helices in subunits (shown from above).

Aquaporins

Two excellent animations showing water transfer through aquaporins can be found on this page.

• http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2003/animations.html

Aquaporins

Some roles of aquaporins in humans:

• Aquaporins 1, 2 and 3 are expressed in the kidney tissue, where they are involved in water reabsorbtion from urine.

• AQP1 is involved with concentration of urine and is expressed in the proximal kidney tubule cells.

• AQP2 is synthesised but not inserted into the apical plasma membrane of collecting duct cells unless ADH stimulates them. Water is then taken up from the lumen of the collecting duct.

• AQP3 is expressed on the basal membrane of collecting duct cells, allowing water transport into the blood when water floods in through the insertion of AQP2 into the apical membrane.

Aquaporins

Disease

• Mutations in AQP2 in humans which result in failed insertion of channels on the apical membrane of collecting duct cells give rise to insensitivity to ADH and produce a form of nephrogenic diabetes insipidis.

• This disease is characterised by chronic urination, dehydration and resultant K+ depletion.

Ligand-gated ion channels

• Ligand-gated channels are opened by a signal molecule binding to the protein.

• The resultant conformational change triggers the opening of the channel.

• Good examples of ligand-gated channels can be found at neuromuscular junctions and at the synaptic junctions between nerve cells.

• This enables a solution to the problem of transmitting an electrically based nerve action potential across the gap between nerves or neuromuscular junctions.

• The nerve action potential is transduced to a chemical signal that can cross the synapse gap, giving rise to an action potential in the adjoining nerve cell or triggering myofibril contraction in a muscle cell.

Ligand-gated ion channels

Voltage-gated ion channels

• Change in polarisation of a membrane results in the opening or closure of voltage-gated ion channels.

• Good examples are the Na+ and K+

voltage channels associated with delivering a nerve impulse along a nerve axon.

• The next few slides show a diagrammatic representation of the propagation of this depolarisation as a result of the action of ligand- and voltage-gated channels.

Propagation of a nerve impulse

Propagation of a nerve impulse

Graph by en:User:Chris 73, updated by en:User:Diberri, [GFDL (www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons

1. Na+ channels open and Na+ flows into the axon, causing depolarisation.

2. Na+ channels close when the action potential is reached. K+ channels open, allowing efflux of K+ to repolarise membrane.

3. Axon becomes hyperpolarised. K+ channels close and the Na+/K+ ATPase pumps potassium back into the axon and sodium out during the refractory period. Sodium and potassium also leak down electrochemical gradients, aiding a return to the resting state.

1 2

3

Propagation of a nerve impulse• A nerve axon is said to have a resting potential when it is not triggered. This is generated by the

action of systems such as Na+/K+ ATPase. This results in a net negative charge on the inside and a net positive charge on the outside.

• The influx of Na+ into a nerve cell down its electrochemical gradient, as a result of the triggering of its associated ligand-gated channel, results in a depolarisation of that local region of the plasma membrane.

• If sufficient Na+ enters the cell the resultant depolarisation triggers the opening of voltage-gated Na+ channels, increasing the depolarisation further and generating an action potential.

• The depolarisation spreads, triggering waves of opening and inactivation of the voltage-gated Na+ channels and propagating the action potential. These channels, once opened, eventually enter an inactive state, preventing dissipation of the gradient or further triggering by neurotransmitter ligands.

• K+ voltage-gated channels also react to the depolarisation but in a delayed fashion.

• They are triggered to release K+ back across the membrane to balance the Na+ influx.

• This helps to repolarise the membrane, triggering the resetting of the voltage-gated Na+ channels to the closed state, which is receptive to neurotransmitters. On repolarisation the K+ channels also close.

• The distribution of Na+ and K+ is re-established by Na+/K+ ATPase and by leakage down electrochemical gradients. The action potential is therefore spread unidirectionally along the axon.

Propagation of a nerve impulse

• A good animation of the role of voltage-gated sodium channels in propagating the action potential along an axon can be found on this page.

• http://www.youtube.com/watch?v=ifD1YG07fB8