Membrane Transport Notes
Outline HERE
Transport Across Membranes

One important feature of the structure of biological membranes is that the lipid bilayer forms an effective barrier to most polar substances, which includes most cellular foodstuffs and ions. Consequently cells have evolved many mechanisms for transporting important polar molecules into and out of cells. One exception to this rule is water. Despite its polarity, water penetrates lipid bilayers, necessitating cellular systems for controlling osmotic pressure.

Most molecules of biological origin must be transported across cellular membranes in a process that involves specific, specialized transport proteins. Transport of molecules across membranes comes at a price (protein synthesis, energy (in some cases)), but affords the cell virtually complete control of what enters and exits from it.

Energy considerations

Everyone is familiar with the process of diffusion. If one places a drop of dye into a glass of water, it will eventually spread out evenly throughout the glass. This is the due to the process we call diffusion. The natural tendency of materials in solution is to diffuse and remove concentration gradients. Movement of materials against a gradient (visualized as "pumping" a substance from a low concentration to a region of even higher concentration) comes at a price - it requires energy. Conversely, movement of materials with a gradient (from a region of high concentration to a region of low concentration) is energetically favored. The free energy difference for the concentration differences for uncharged molecules is given by

G = RTln(c2/c1)

where the molecule is moving from concentration c1 towards concentration c2.

For charged species, the charge difference must be taken into account, as follows,

G = RTln(c2/c1) +ZFV

where Z is the electrical charge of the transported species, V is the potential in volts across the membrane, and F is the faraday constant. See also HERE.

Types of transport

There are two basic types of transport systems for moving substances across membranes:

1. Passive/facilitated transport - driven solely by diffusion
2. Active Transport - drive by other energy sources

Passive/facilitated transport can occur simply as the result of the process of diffusion (passive) or may be assisted (facilitated) by specific molecules that enable the transfer of substrate across a membrane. In either case, the end result would be equal concentrations of the transported material on either side of the membrane, if nothing else were done. A major difference, however, is that facilitated transport speeds achievement of the equilibrium dramatically. Facilitated transport of glucose across a membrane can be 1000 to 10,000,000 times faster than passive transport of the same molecule.

Active transport processes usually work against a concentration gradient, pumping molecules from a lower concentration on one side of the membrane to a higher concentration on the other side of the membrane. This is opposite the direction of diffusion and consequently requires energy to occur. In some cases, the gradient can be formidable. Active transport mechanisms employ specific protein molecules variously called carriers, permeases, porters, translocases, translocators, and transporters.

Cells expend energy in order to build concentration gradients of ions (for example, Na+ and K+ as described below). This electrochemical potential gradient represents energy that can also be utilized for the purposes of the cell. As we shall see later, mitochondria harvest energy from a proton gradient they create in order to make ATP in oxidative phosphorylation. Energy from electrochemical potential gradients is also used by the cell to drive transport of molecules across membranes (see Na+/glucose below).

Each of our cells transports ions such Ca++, Na+, and K+ against a concentration gradient. Transporting "against" a concentration gradient (pumping) means that the ion is being moved from an area of lower concentration to a area that has a higher concentration. It is called "against" because the constant pressure due to diffusion goes in the opposite direction.

Na+/K+ ATPase System (active transport)

The Na+/K+ ATPase System (also called Sodium-Potassium Pump) of plasma membrane is an ion pump. It functions to transport 3 Na+ ions out as it transports 2 K+ ions in coupled to the hydrolysis of one molecule of ATP. Coupling of this transport to ATP hydrolysis is essential. By doing so, the energetically unfavorable reaction of moving Na+ and K+ ions against a concentration gradient is converted to an energetically favorable reaction. The Na+/K+ ATPase system is an electrogenic reaction (electrogenic = transport system that results in a net change in charge) which functions in all animal cells to control their osmotic environment. Nerve cells use the Na+ (conc. higher outside than inside cell) and K+ (conc. higher inside cell than outside) gradients for several transmitting signals. The high concentration of Na+ created outside the cell by the Na+/K+ ATPase System is used by the Na+/glucose transporter to 'carry' glucose into the cell. The Na+/glucose transporter is an ACTIVE transport system, because it can move glucose from low concentration to high concentration, thanks to the energy provided by the high Na+ concentration outside the cell and low concentration inside the cell. Cells use the Na+ gradient to their benefit, by allowing Na+ to flow inwards, stimulating Ca++ pumping outwards (via the Na+/Ca++ exchange pump). An important heart drug, digitoxigenin, blocks the Na+/K+ ATPase System and leads to high Na+ concentration inside of the cell. This, in turn, causes Ca++ to increase (since the Ca++ cannot be pumped OUT using the Na+/Ca++ exchange pump), causing the heart to pump more energetically.

Transport systems that move one molecule one direction and another molecule in the opposite direction (example = Na+/K+ ATPase System) are known as Antiports. Transport systems that move two molecules in the same direction (Na+/glucose transporter) are known as Symports (also called Synports in some books).

Note that the Na+/K+ ATPase System requires ATP to pump against the gradients. The process is reversible. If reversed (pump Na+ in, K+ out), the pump system could generate ATP, but rarely does because to do so for very long would upset the osmotic balance of the cell. Students should note, however, that running pumps backwards is a potential source of energy that in some cases is employed by the cell.

Ca++ ATPase (active transport)

Calcium is an ion that cells must regulate the concentration of (noted last term). Ca++ is important for muscular contraction, so muscle cells sequester stores of it in the sarcoplasmic reticulum. Release of Ca++ from the sarcoplasmic reticulum causes muscular contraction. Muscular relaxation requires the quick transport of calcium BACK into the sarcoplasmic reticulum. The Ca++ ATPase is a protein in the membrane of the sarcoplasmic reticulum that facilitates the movement of calcium back into storage. Eversion is a mechanism commonly used by transport proteins to shift the opening from one side of the protein to the other after the molecule to be transported is bound. Note also in the mechanism that a phosphate from ATP gets transiently transferred to the Ca++ ATPase (step 2). The attachment is to an aspartate residue in the protein and this phosphoaspartate intermediate is common among a group of ion transport pumps known as P-type pumps.

P-type Pumps (active transport)

P-type pumps (or P-type ATPases) are common transport systems in cells. Analysis of the yeast genome reveals at least 16 such proteins. Two move calcium. Others move sodium/potassium or metals, such as copper. Five transport phospholipids with amino head groups from one side of the lipid bilayer to the other (transverse movement). Transport systems that move phospholipids across the bilayer are known as flippases.

Multidrug Resistance Protein and CFTR (active transport)

Another important class of transport proteins contain an ATP-binding cassette (ABC) domain. Examples include the multidrug resistance protein (MDR), the cystic fibrosis transmembrane conductance regulator (CFTR), and the histidine permease of S. typhimurium. MDR was originally identified in cells that lost their sensitivity to drugs. Expression of MDR appears to provide resistance to many drugs and operates by pumping many drugs outside of cells before they can exert their effects. CFTR is a protein that, when mutated, gives rise to cystic fibrosis. CFTR is a an ATP-regulated chloride channel that, when blocked, causes dehydrated mucus to accumulate in lungs. The histidine permease acts to transport histidine into the bacterium. All of these proteins have two common ATP binding domains and two membrane binding domains. There are 79 ABC proteins in E. coli. As with other ATP utilizing transport proteins, hydrolysis of ATP generates conformational changes in the protein that ultimately result in movement of the desired molecule across the membrane.

Secondary Transporters (active transport)

Other active transport systems use energy from a source besides ATP to move molecules against a concentration gradient. The energy source here is partly that of diffusion and (often) partly that of the charge difference across a membrane. Note that diffusion CAN be used in an active sense if, in the process of diffusing from a high concentration to a low concentration, the molecule CARRIES WITH IT another molecule from a low concentration to a high concentration. A good example described above was the Na+/glucose system. Here the higher concentration of Na+ outside the cell is used to CARRY glucose into the cell AGAINST a concentration gradient of glucose. That is, glucose is moved from low concentration to high concentration by the Na+ gradient. Another similar example is that of the lactose permease pump of E. coli. In this system, a proton gradient is used to carry a lactose against a concentration gradient. Note that all of these secondary transport systems REQUIRE proteins that specifically bind the molecules described here and that they evert as a means of moving the molecules from one side to the other.

Ion Channels

Ion channels are PASSIVE transport (facilitated, actually) systems for moving ions across membranes. Ion channels (also known as ionophores) can allow molecules to move as fast as diffusion will allow (about 1000 times faster than a pump). Key features of ions channels are

Ion channels are used in the transmission of nerve signals and other processes of the body.

Neurotransmitters

One of the ways in which the gradient of Na+ and K+ built up by the Na+/K+ ATPase is used is for transmission of signals by nerve cells. Within a given nerve cell, signals are propogated by inflow of Na+ (thanks to opening of the gates of the nerve cell, allowing Na+ to flow in by diffusion) and the outflow of K+ (thanks to opening of the gates of the nerve cell, allowing Na+ to flow in by diffusion). Note that for a cell at rest, the Na+ concentration is HIGHER outside than inside, so Na+ flows IN. Note also that for the same cell, the K+ concentration is HIGHER inside than out, so K+ flows OUT.

Movement of these ions across the membrane causes voltage changes across the cell membrane and this voltage moves along the nerve cell as the diffusion progresses. The first gates to open are the Na+ gates followed by the K+ gates. As the incoming Na+ ions move down the nerve cell, a voltage is propagated all along the nerve cell. At the end of the nerve cell, the signal must be communicated from one cell to another. Note that after the signal moves through the nerve cell, the gates close and the Na+/K+ catches up and reestablishes the gradient for each ion that existed before the signal passed through.

The space separating the individual nerve cells has a name - a synaptic cleft. When the signal reaches the end of one nerve cell, it stimulates the release of a neurotransmitter molecule. One of these is acetylcholine. Notice in the figure above that the signal propagates down the first nerve cell to the presynaptic membrane. At the synaptic cleft, the acetylcholine is released and it interacts with the other nerve cell junction (postsynaptic cleft) at a receptor called the acetylcholine receptor. The acetylcholine receptor is what is known as a ligand-gated channel. When acetylcholine binds to it, it opens, causing Na+ and K+ to enter and leave the nerve cell, respectively. Thus, the second nerve cell begins voltage changes just like the first nerve cell and the signal continues to move down it. Note that these signals can be transmitted extremely fast (on the order of 0.1 msec).

Electric rays are sea organisms with a tremendous number of acetylcholine receptors that they use to generate voltages of as much as 200 volts to stun prey. It is worth noting that some snake venoms are neurotoxins. Cobratoxin, for example, is a small protein that binds tightly to acetylcholine receptors and prevent the transmission of signals from nerve to muscle.

Sodium and Potassium Channel Proteins

The proteins that form the gates that open/close to allow sodium/potassium to move or not move across the membrane are NOT controlled by ligands. Instead both proteins appear to be sensitive to voltage changes. Remember at the beginning of the transmission of the nerve signal at the post-synaptic cleft that a voltage change is initiated by movement of K+ and Na+ ions. As this voltage change moves down the axon of the nerve cell, Na+ and then K+ gates open in response to the voltage change. Opening of Na+ and K+ allows more Na+ in and more K+ out. Thus, as the signal moves DOWN the nerve cell, it is kept alive by inflowing Na+ and outflowing K+. The Na+ gate protein and K+ gate protein are similar in general structure. The Na+ protein is VERY tightly bound by tetrodotoxin, a neurotoxin from puffer fish. A lethal dose of tetrodotoxin is about 10 nanograms (!).

Selectivity of Channel Proteins

It is essential for gate proteins to be able to selectively allow desired molecule to pass, while not allowing undesired ones to pass. As you might expect, one of the keys to this selectivity is geometry. Geometry, however, cannot be the only mechanism of selectivity, however, for how would a channel selecting for a larger ion (K+) exclude smaller ions, such as Na+? The answer lies in the chemistry/geometry of the hydration of these two ions.

As ions pass through the K+ channel, they must have the water molecules stripped from them. The K+ gate has carbonyl groups positioned so as to favor the dehydration of K+, but not Na+. Since dehydration is essential to pass through the channel and Na+ is not easily dehydrated, it is rejected, whereas K+ is dehydrated and is allowed to proceed.

Gap Junctions

In addition to ion channels, which are very selective for ions entering/exiting the cell, other channels BETWEEN cells (in multicellular organisms) exist to permit the movement of molecules. Gap junctions are made of a protein called connexin and are fairly non-selective in what they allow to pass through them. Small ions and non-polar molecules less than about 1000 in molecular weight can pass through them. The primary exclusion mechanism here is size. Proteins, DNAs, and large carbohydrates are too large to pass through gap junctions, but simple sugars, ions, etc. can pass readily. Gap junctions can be closed by Ca++ and H+, permitting cells to prevent the importation of undesirable molecules from other damaged cells.