Lecture 32

Membranes III

Last time we finished describing (briefly) the Na+/K+ pump, which was used to maintain a gradient of Na+ against the concentration gradient.The complete cycle of the Na+/K+ pump, an antiport, looked 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.

A scheme of the process is here, with high energy transporter (enzyme) forms in red borders:

As we said, we need this Na+ and K+ gradient for two things. First, we can use the Na+ gradient to our favor (favorable chemical potential) to bring into the cell other solutes if we associate the process with bringing in Na+ to the cell. Second, these processes increase the Na+ concentration in the cell, and if we don't pump it out, osmotic pressure would burst the cell apart.

One of the process associated with Na+ concentrations is Ca+2 uptake in muscle cells, which is an Na+/Ca+2 antiport. When we have the Na+/K+ working properly, the Na+/Ca+2 system is OK. If we inhibit the Na+/K+ pump, which pump Na+ out of the cell, the Na+/Ca+2 pump starts working overtime to get rid of the Na+. On the process, it bring in Ca+2, wich triggers muscle contraction, particularly in cardiac muscle. The result, your heart beat goes up. This is the priciple of operation of two drugs, digitoxin and oubain:

There are two drugs/poisons bind strongly to the outside binding site of the Na+/K+ pump, inhibiting its operation. In small doses, the two molecules are used to treat patients with cardiac conditions.

Ca2+ pump

Another important pump system is the Ca+2 pump (or Ca+2ATPase). For what we saw above, regulating the Ca+2 concentration in the cytosol is important, because Ca+2 is crucial as a signaling agent in the regulation  of several metabolic processes, including muscle contraction. The concentration of Ca+2 in the cytosol is kept 4 times smaller than the one in the extracellular fluid, and other processes (i.e., muscle contraction) will bring Ca+2 inside. We thus need a pump to maintain this 4 fold excess in the outisde, and as usual, we will be fighting against the concentration gardient.

The transporter exists also in two states, E1 and E2, one which has affinity for Ca2+, and the other one which does not. The process is sumarized as follows:

- In the first step, Ca+2 binds to the transporter, and this promotes binding of ATP and phosphorylation of the protein (formation of the E1-Ca+2-P complex).

- Phosphorylation of the protein makes a high energy form of the protein, wich triggers a conformational change of the protein (E2), exposes the Ca+2 binding site of the protein to the extracellular fluid, and promotes release of Ca+2 in the outside. This step generates a low energy E2-P complex.

- The phosphate is hydrolyzed from the E2-P complex, again triggering a conformational change, and we regain the E1 form of the transporter, which is ready to bind more intracellular Ca+2.

At this point we are at the begining of the cycle once again. A scheme that represents this is shown below, with high energy forms of the transporter shown with red borders:

There are many more active transport pumps, to many to put here and analyze in detail. In all this pumps, transport (be it symport, antiport, or uniport) the important thing is that the energy consuming step (usually phosphorylation of the pump and hange in conformation) is directly connected with the transport of the solute. They are therefore called primary active transport. There are other active transport processes that use something made by a primary active transport process: The ion and charge gradient.

Ion gradients - secondary active transporters

While all this pumps do what they gotta do, we are generating something that we can use in our favor: A chemical gradient (things get more concentrated at different sides of the membrane), and an elecric gradient (a voltage) across the cell membrane. As we saw before, this voltage is proportional to the charge (the concentration of ions) we have, and will work in our favor - Ions will move along this electric potential gradient.

As we mentioned, we can use this gradients to couple the transport of another solute with it, in what is known as secondry active transport.

Lactose permease

A good example of an ion gradient driven pump is lactose permease, which is used in E. Colito accumulate sugars in the cytosol. The ion gradient in this case is a H+ gradient, and lactose permease cotransports H+ and lactose into the cell. The H+ gradient is generated by metabolic processes, which increse the [H+] outside the cell.

The transporter has two forms, E1 and E2 (duh). E1 has a low affinity lactose binding site facing the interior of the cell. The E2 form has a high affinity binding site facing the extracellular fluid. The E1 form can only convert to the E2 form when the H+ and lactose binding sites are either both filled or both empty.

- With the enzyme in the E2 form (high affinity for lactose), the transporter binds lactose. If the [H+] in the outside increases, then the conformation of the transporter changes, fliping the binding site to the inside and making it low affinity (E1). Lactose is then released.

- Metabolic processes decrease the [H+] concentration, and E1 (which until now had H+ bound) looses H+, and since both binding sites are empty, we changes again to the E2 form, with the high affinity binding site pointing to the outside.

The process is now repeated, increasing the internal concentration of lactose in the cell. the process is summarized in the following scheme:

Ion selective channels

We have another way of transporting ions across membranes apart from pasive and active transporters. These are called ion channels, and in contrast to normal transporters, they do not show solute saturation - They act basically as 'gates' that allow for free access for certain ions into and out from the cell. These channels can be activated (opened or closed) by very small variations of the concentration of certain signaling molecules. The opening of the channel is rapid, and allows a large flow of ions along the concentration gradient very fast.

The best studied ion channel is the acetylcholine receptor in the synapses of neurons, which is responsible for the transmition of the electric impulses along neurones than make us move, think, see, hear, etc., etc.

An electric signal carried along a neurone (which is nothing more than a Na+ gradient diffussing along the neurone) reaches the synapse, and acetylcholine (acetylCoA) kept in the cell is released into the cleft between the two neurons:

The acetylCoA molecules reach the other neurone fast by diffusion, and they bind to the acetylcholine receptor in the membrane. When the molecule binds to the channel, there is a conformation change which opens a pore which has the right size for Na+ and K+. When there is no acetylCoA around, the channels are shut.

Since we have an excess of Na+ in the outside due to the Na+/K+ pump, Na+ in the cleft between the two neurons rushes into the postsynaptic neuron, and the signal is then transmited.

One important thing about ion channels is that their 'switches' (activators, acetylCoA in this case) are transient - The channel oly opens for a short period when acetylCoA binds, and it closes very rapidly afterwards. Otherwise, we would have an information overload! This is what some poisons do - When you get bit by a snake, its neurotoxins (peptides that look like acetylCoA, but bind irreversibly to the acetylcholine receptors) bind to the acetylcholine receptor and they leave the channel open. Then come muscle spasms, respiratory failure, cardiac arrest, and all that ugly stuff.

If you read the whole thing, and keep in mind that there are many more pumps and transport systems than the ones we saw here, you will appreciate how complicated and perfectly balanced the cell is, and therefore how specialized the membrane has to be. Transport of certain ions creates ion gradients, which are used in cotransport of other solutes (nutrients), signaling (Ca2+, Na+ in neurones), and at all times, we have to keep the balance in order to avoid blowing the cell up. Pretty nifty...

Prepared by Guillermo Moyna, 1999.