Membranes II

Last time we described membrane composition, membrane proteins, and wraped-up by mentioning the fluid mosaic model. Today we will start investigating transport of solutes accross membranes. We'll talk about the thermodynamics of the process and we will also see several examples of different transport proteins.

Transport accross membranes

As we said, all but one polar solute are transported accross membranes using transport proteins, and most transport proteins are integral transmembrane proteins (except for ionophores - See below). In order to understand how these molecules make transport possible, we have to analyze first the thermodynamics of the transport across membranes.

Passive transporters

Say we have a jar filled with water and with a permeable membrane cutting it in half. If we add salt (NaCl) on one side, the concentration of NaCl on this side (C₁) will be a lot higher than on the other side (C₂), at least initially.

However, after a while the solute in the left will permeate to the solution in the right - This is intuitivelly what we figure out will happen, and it is what happens. We can also think of it thermodynamically. By having more solute in one side, we are going against entrppy (we are ordering things to one side of the membrane), and things will try to equilibrate. We can think of the process as the following reaction:

Where A(left) and A(out) are the same solute, but at different concentrations:

We can treat this system using thermodynamic equations. The DG is defined in the same way that the DG for any other reaction, but now we only have the concentration differences:

If you think a little, it will take you no time to figure out that the DG^o for this reaction is (has to be) zero. Therefore:

This quantity is called the chemical potential, and it tells us how much free energy we can get from equilibrating the concentration of both solutions. The 'reaction' will stop when C₂ / C₁ = 1, and then DG will be 0. Before that point, DG will always be < 0, and there is always a tendency for the solutions to equilibrate. We can always use the chemical poteintial to our favour, because it is favourable to move in the same direction than the chemical gradient.

This was for a permeable membrane, such as a dyalisis bag, and is called simple difussion. In a biological membrane, this type of unasisted difussion, or non-mediated transport, will only happen for non-polar solutes (O₂, CO₂, cholesterol, small hydorphobic molecules and peptides). A polar solute on one side of the membrane cannot go through the membrane, because it cannot pass through the non-polar lipid core of the membrane. Not only this, before the polar solute goes into the membrane, we will have to srtip it of all its hydration water, which is energetically unfavourable. The free energy profile for this would look like this:

In the same way that a chemical reaction had a DG of activation, going through a membrane will have a large positive DG for simple diffusion. In order to help in the diffusion in the direction of the chemical gradient we use integral membrane proteins called transporters, permeases, or porins, that basically lower the activation energy of the passage through the membrane. Instead of having to contact the membrane non-polar fatty acids, the solute is in contact with the inner side of a transmembrane protein that makes a transmembrane channel (a 'tube') of higher polarity:

The mechanism is parallel to that of enzymes: the solute will bind the outer side section of the porin, and the favourable non-covalent contacts (DG_binding) will lower the increase in energy due to the loss of solvation (DG_solvation). Through the pore, there will be favourable non-covalent interactions between the solute and the protein's transmembrane channel that lower the free energy (they lower the energy that represents being 'bare', with no water around it, sort of like substituting the water-solute interactions). These interactions are between polar residues of the protein and the solute. On the other side, the solute regains its solvation shell and goe into the cytosol (if the transport was from the extracellular fluid to the cytosol.

If we compare the rates of simple and facilitated difussion, we will see that the process follows Michaelis-Menten kinetics. The reaction in this case is:

The initial rate of transport versus solute concentration in the outside (S_out) measured while S_in is negligible is the infamous hyperbola:

Here K_M is replaced by K_T. In the same way that we experience saturation with enzymes, we will experience saturation with transporters: After a certain [S]_out, the rate will remain constant because the pore is saturated with substrate.

Furthermore, in the same way that enzymes are very specific for their substrates, transporters are very specific for the solutes they pick - The same principles apply: The more favourable interactions we get between solute and transporter, the best we can counter balance the loss of solavation, lowering the free energy of the passage through the membrane.

A good example of facilitated diffusion is glucose permease in erythrocytes. First, the K_T for D-glucose is 1.5 mM - The same permease has K_T of only 20 and 30 for D-mannose and D-galactose, in which only a couple of hydroxyl groups have different stereochemistry to D-glucose. For L-glucose, the K_T is 1000.

This type of transport in which we move solutes in the same direction than the chemical gradient (in favor of the chamical potential) is called passive transport or facilitated difussion. It is carachterized by increase in the rates of diffusion down a concentration gradient and high specificity for the solutes.

Another thing that can be generalized for transporters is that they alternate between two conformations. In one, the side that will bind to the substrate is exposed to the aqueous environment around the mebrane. One the solute binds, there is a change of conformation that pushes the solute through the pore and, more or less shuts the side that was 'open', and opens the protein pore on the other end of the membrane. Here the solute goes back into solution:

There are several drugs that are basically transporters. As we will see below, in most cases cells will try to maintain a gradient of ions between the cytosol and the extracellular fluid. This means, the concentration of, for example, [Na⁺] inside the cell is 12 mM, while outside it is 145 mM. The reverse happens to [K⁺], which is 4 mM in the outside and 140 in the inside. There is a large chemical potential for both ions which would try to equilibrate them - However active transportes (see below) and the membrane keep things the way they are.

Two antibiotics, Gramicidin A and Valinomycin screw this up. Gramicidin A is a 15-residue peptide with alternating D- and L-amino acids that dimerises putting both formylated N-termini together, and spans right across the membrane. In doing so, it makes a small pore in the membrane through which K⁺ and Na⁺, can escape to the extracellular fluid or get into the cytosol through facilitated diffussion:

Valinomycin is a small cyclic hexapeptide peptide with several valine residues or derivatives of valine. We also have alternation of D- and L-amino acids. Valinomycin is not as big as Gramicidin A, so it cannot form a pore. Instead, it can bond tighlty to K+ ions by coordination with the peptide group carbonyl. When Valinomycin binds K⁺, all the polar oxygens turn towards the inside, and all the hydrophobic valine-like residues point to the outside. This makes the antibiotic higly liposoluble, and it travels through the mambrane real fast. Once in the inside (or outside), it releases the K⁺, and goes back to a conformation in which the carbonyl groups are involved in h-bond with water.

Both Gramicidin A and Valinomycin are called ionophores, molecules that love ions. They screw up the ion gradients in the cell, and as we will see, this has a devastating effect in the regulation of cellular processes, killing the cell.

Now, back to porins and transporters. In some cases of transport we can have more than one solute going in or out. We can also have one going in at the same time that another one goes out. If we have only one solute being transported, we call it uniport. If we have two going the same way, we call it symport. If one goes one way, and the other the other way, we call it antiport. This applies for transporters as well as for active transport proteins (or pumps), described below and next class.

Active transport

Many times (most of the times) going down the concentration gradient is not enough, and we have to move in a solute even if its concentration inside is already higher than in the outside. Not only we'll be going against the membrane and we will have a transport free energy barrier, but to this we will have to add the free energy of the chemical potential, which will be against us in this case.

This type of transport is called active transport, because the membrane protein is active against the chemical gradient. It requires consumption of energy in the form of ATP (the processes are coupled to ATP consumption), and in most places you will see people refering to these proteins are ATPases. One of the calsical ATPases is the Na⁺/K⁺-ATPase, also known as the Na⁺/K⁺ pump.

This pump performs the following reaction:

In this case, since we have two positive charges entering the cell and two leaving, we also generate an electrical potential across the membrane. The free energy equations are now:

where ZA is the ionic charge of the solute, F is the Faraday constant, and DY is the electric potential difference between in and out. If this pump does not work, other processes would make the [Na⁺] concentration in the cytosol very high. This would create a large osmotic pressure difference, because water wants to get in the cell to equilibrate the [Na⁺] to that of the extracellular fluid. This influx of water into the cell would cause the cell to swell, and eventually burst.

How does it work? The first step is the phosphorylation (by ATP) of a key aspartate residue. This residue is phosphorylated only when the protein is bound to Na⁺. The hydrolysis of this phosphate group only occurs when the enzyme is bound to K⁺. So, the pump has two conformational states, E₁ and E₂, which have different structures, catalytic activities, and specificities for substrates.

The complete cycle of the Na⁺/K⁺ pump looks like this:

- The transporter in the E₁ (high affinity for Na⁺) state binds three Na⁺ in the cytosol, and the aspartate residue gets phosphorylated, forming a E₁-3Na⁺-ATP complex.

- Hydrolysis of ATP generates ADP and a high energy E₂-3Na⁺-P complex, which relaxes by chainging its conformation, exposing the Na+ binding site to the outside. This is followed by release of three Na⁺ and generation of a E₂-P complex, which has high affinity for K+.

- The pump now binds two K+ ions from the extracellular fluid, to form the E₂-2K⁺-P complex.

- Hydrolysis of the phosphate generates a high energy E₁-2K⁺ complex.

- This complex releases energy by changing its conformation, exposing the K⁺ binding site to the cytosol, and releasing two K⁺ ions. In the process, the E₁ form ot the pump is regenerated, and the process starts over.

Prepared by Guillermo Moyna, 1999.