Barriers, Pores, Pumps and Gates

The areas covered in this plenary are

.....if you haven't previously studied Biology, you may find the following useful

In the beginning....

in the beginning

life ascending

Before there were living things on the planet there were self-replicating molecules that floated around in a primeval soup busily replicating. Isolated molecules have no internal environment and so are unable to indulge in homeostasis and, from a physiologists viewpoint, are therefore not alive. However, at some point these self-replicating molecules 'discovered' a way to isolate themselves from their immediate environment. If you live in a watery environment and you want a little isolation, a fat bubble is what you need, because fat or oil and water don't mix, so a fat or lipid layer will act as an effective barrier to the outside world. Once contained within a fat bubble the self-replicating molecules had an internal environment that they could regulate to suit themselves. At this point we can pass over several hundred million years of evolution and call these thing 'cells'. By this simplistic definition, a cell is a collection of self-replicating molecules (DNA) surrounded by a fat bubble (the plasma membrane). As a definition, it will do for now.

If you are interested in reading a brilliant book about the very latest ideas on the origin of life (and other things), try Nick Lane's book, "Life Ascending: The Ten Great Inventions of Evolution".

Channels and pores.

sodium channelA simple, completely exclusive barrier between the intracellular and extracellular environments is not by itself much use in homeostasis. The barrier with the outside world must allow in those things necessary or growth and development whilst excluding everything else. In short it must be selectively permeable. Pure lipid would be practically impermeable to most water-soluble substances so a plasma membrane contains channels and pores built from protein molecules to enable selected substances to enter (or leave) the cell. A pore or channel is a protein with a hydrophobic (water hating, lipid loving) exterior which can sit happily in the membrane and a hydrophilic (water loving) centre through which water and small water soluble molecules can pass. If such a molecule is inserted into a plasma membrane so that one end sticks out of the cell and the other end sticks out into the cell interior water soluble compounds can cross the membrane without ever really coming into contact with the lipid . A plasma membrane almost always includes channels that allow water, K+, Cl-, HCO3- Ca2+ and Na+ to cross.

In general then, O2 and CO2 and small, uncharged polar solutes such as urea or ethanol can diffuse into and out of cells across the lipid of the plasma membrane. For all practical purposes, water and ions can only diffuse in and out of cells through a channel or pore.

Evidence to date indicates that water pores are always open (See "Why do we need kidneys?" for some idea about how cells can change their water permeability), but ion channels have the vitally physiologically important ability to CLOSE and let nothing through. Neurones (nerve cells), for example, have masses of Na+ channels, but at rest, practically zero Na+ conductance because the Na+ channels are all closed. Ion channels are present in all plasma membranes but they will be open or closed depending on the needs and the function of the cell (See "Getting the message across" or this Review for more).

Aquaporins.

In real life, channels and pores are much more than watery holes in the membrane. They perform incredible biophysical feats to allow their "chosen" molecule to permeate whilst excluding all others. Aquaporins, water channels, are a shining example of cleverness. Each aquaporin (AQ) molecule has 6 membrane spanning regions that fold back on one another to form a pore. A functional water channel is made up of 4 identical aquaporin molecules (the channel is therefore a homotetramer; 4 the same). Aquaporins somehow allow a relatively huge water permeability and a very low proton (H+) permeability which is particularly clever because protons can pass through water molecules! See the Theoretical and Computational Biophysics Group for an idea of how they accomplish this. There are literally dozens and dozens of aquaporin isoforms (the same but different). The main ones are numbered 1 to 10; The salivary glands have AQ5, the small intestine AQ7, the colon AQ3, AQ4 & AQ8. The kidney has AQ's 1-5 & AQ6 (Don't take this list too seriously; this is an area of active research and more aquaporins are found daily!) There are things to read if you are interested in the basic biochemistry of aquaporins or, if you are really keen, a detailed review of the physiology of aquaporins. Alternatively, you could read up on the discovery of aquaporins (These last two links should work from University computers, but might not from elsewhere).

Diffusion and the electrochemical gradient

Channels and pores can exclude a molecule from the cell or keep something inside the cell by being closed (or by not being present, i.e. Na+ cannot cross the cell membrane if it does not contain Na+ channels). If the channel is present and it is open, then the permeating substance can pass through the channel down its concentration gradient, from a region of high concentration to a region of low concentration (diffusion).

nernst equationIf the substance crossing the membrane is charged (e.g. an ion such as K+ or Na+) and there is an electrical potential difference across the plasma membrane (which there is) then the electrical potential will also drive the substance across the membrane. The chemical driving force (of the concentration gradient) and the electrical driving force (of the membrane potential) add algebraically to give the electrochemical driving force. The electrical and chemical components of the electrochemical gradient can operate in the same direction to create an electrochemical gradient that is the sum of both of them. Or, if the concentration (chemical) gradient is pushing one way and the electrical gradient is pushing the other way then they cancel each other out either partially or completely (the electrochemical gradient is zero). It follows that for every permeable ion there is a membrane potential, which will exactly balance the concentration gradient for that ion. This membrane potential is known as the equilibrium or Nernst potential and it may be calculated using the Nernst equation.

k lossJust to make things really complicated, it is the diffusion of ions that creates the membrane potential in the first place. Imagine a cell membrane permeable only to K+ and further imagine that there is a 10 fold K+ gradient running from the inside of the cell (100 mM) to outside the cell (10 mM). Potassium will diffuse down the concentration gradient and out of the cell. However, every time a positively charged potassium ion crosses the membrane it makes the inside of the cell membrane a little more negatively charged (The positive charge is being lost to the cell. This negative charge will try to attract positively charged K+ back into the cell, or at least try to prevent further K+ loss). The build up of negative charge on the inside of the membrane is the membrane potential. Assuming that no other ion can cross the membrane, the potential will build up until it acts as a driving force exactly equal and opposite to that of the chemical gradient. The cell membrane potential will be exactly equal to the equilibrium potential for potassium. At this point there will be no further net movement of potassium.

If any ion other than potassium is allowed to cross the membrane then the membrane potential will also depend on the concentration gradient (and the relative permeability) of that ion. What doesn't cross the membrane is, in fact, much more important in generation of the membrane potential than what does. A significant proportion of the anions (negatively charged ions) inside the cell are proteins. Proteins cannot ever cross membranes, their membrane permeability is zero. Most cells, most of the time, have a very low Na+ permeability and a high K+ permeability therefore K+ leaves the cell and the two most obvious ways of counteracting this build up of charge, namely, loss of anion or influx of cation (positively charged ion, e.g. Na+) are prevented by the properties of the membrane. A simple and useful way of thinking about the membrane potential is to consider that it is created by K+ movement and that it can then drive the movement of other ions. This is nearly true for most cells. The best way to find out a more about the Nernst and Goldman (Goldman is the Nernst equation's big brother which takes into account the movement of more than one ion) is to play with them online. Change the values for K+ concentration and see the effect on membrane potential. To see this program at its best, choose 'Model' from the top menu bar and then 'Action Potential' from the presets menu. Alternatively, you could read the help pages.

There can be no net movement of a substance through a channel or pore

against its electrochemical gradient.

Active Transport

primary active transportCells do move substances across the plasma membrane against their electrochemical gradients, not through channels or pores but by active transport. Moving against an electrochemical gradient requires an input of energy in order to overcome the gradient (just as a car can free wheel down hill but needs an engine to get back up again). Active transporters are enzymes that utilise metabolic energy in order to 'pump' against an electrochemical gradient.

Primary active transport

Perhaps the most straightforward type of active transport is primary active transport in which metabolic energy in the form of ATP is directly utilised by an enzyme to move a substance across a membrane. The most ubiquitous and widely known example of primary active transport process is the sodium pump, or sodium / potassium ATPase. The Na+ pump uses ATP to extrude Na+ from the cell in exchange for K+ (3Na+ out for 2K+ in). The Na+ pump is therefore responsible for the high K+ concentration found within cells and thus the inside to outside directed K+ gradient that generates the membrane potential. So, although the Na+ pump does not itself create the membrane potential (which is a commonly held misconception) it does maintain it by keeping the intracellular K+ concentration high. There are other actively transporting ATPases. A proton pump pumps protons (H+) out of the cell (sometimes in exchange for Na+). Various different cell types utilise a proton pump, in particular the cells in the stomach which secrete gastric acid and kidney cells which are (partly) responsible for regulating the pH of the whole body (and not forgetting the mitochondrial proton pump and its key role in energy production). The calcium pump is another important ATPase that is present in one form or another in all cell types. Intracellular calcium concentration is used as an on/off switch for lots of cellular processes. Increase intracellular calcium concentration and lots of Ca2+ -dependent enzymes get activated. Reduce intracellular calcium concentration and they all switch off again. The Ca2+ -ATPase has a very important part in this process because it is able to maintain intracellular calcium concentrations at very, very low levels.

The ATPases responsible for primary active transport exist in a variety of different forms, but under physiological conditions the only substances that cross the plasma membrane as the direct result of primary active transport are Na+, Ca2+, K+ and H+. If there is such as a thing as a Cl- pump, then no one has found it yet!

Even though there are very few substrates for primary active transport, there are nevertheless active transport processes for a bewildering variety of substances. Everything else that must be actively transported across the plasma membrane moves by secondary active transport.

Secondary Active Transport

secondary active transportWhereas primary active transport makes direct use of metabolic energy in the form of ATP, secondary active transport uses metabolic energy indirectly in the form of a concentration gradient created by a primary active transport process. In other words...... As a direct result of the activity of the Na+ pump intracellular Na+ is low relative to extracellular Na+. There is therefore an inwardly directed Na+ gradient. Just as K+ is keen to leave the cells, so Na+ is desperate to enter. But whereas the cells allow K+ to leave (and thus create the membrane potential) most plasma membranes are practically inpermeant to Na+. So Na+ can't get in, at least not by itself. Secondary active transporters allow Na+ to enter the cell only if accompanied by another substrate. Thus a glucose transporter carries glucose and Na+ into the cell (please note that the Na+-dependent glucose transporter is found in intestinal and renal epithelial cells, other cells have different glucose transporters), amino acid transporters carry amino acids and Na+ into the cell. In effect, the co-transported substrate gets a free ride on the Na+ gradient. So long as the Na+ gradient is bigger than the gradient of the co-transported substrate, whatever it is, will be concentrated inside the cell. This sort of arrangement can work with many different pairs of substrates, but it works especially well with Na+ because the Na+ pump gets rid of the Na+ as soon as it enters the cell and thus preserves the large inwardly directed Na+ gradient.

I've made this introduction to secondary active transport as simple and as brief as I possibly can. The purpose of this paragraph is to get across concept rather than detail. If you are interested in detail, then have a look at this review by Kato et al or see my notes on the proximal tubule. The proximal tubule and the small intestine both use a very similar set of transporters to achieve essentially the same job.

In secondary active transport the Na+ gradient is inwardly directed.

Sodium always, always, always moves into the cell

cotransportThis sort of secondary active transport is known as co-transport because two substances (Na+ and something else) are co-transported together. More properly, it is an example of symport because the two substances are moving in the same direction. Another variant of secondary active transport is antiport where the two substrates move in opposite directions. Sodium proton exchange is an example of this. Sodium moves into the cell in exchange for H+ leaving the cell. The Na+ gradient is the most common driving force for cotransport, but it is not the only one. Some cells have an outwardly directed Cl- gradient that they use to drive HCO3- into the cell

An individual cell, whether part of an organism or a single cell creature, is able to create and maintain an internal environment by virtue of possessing a lipid based plasma membrane which regulates contact with the extracellular environment. Regulation occurs via channels, pores and transporters. The whole process is driven by metabolic energy in the form of ATP that is utilised by the transport processes either directly as in primary active transport or indirectly as in secondary active transport.

All cells regulate their ionic environment; organs, such as the kidney regulate the extracellular environment. There is no such thing as a single or even a "typical" intra- or extra- cellular environment. The precise salt and water balance in a particular cell type or extracellular body fluid compartment is whatever best suits the function of that cell type or body fluid compartment (after all, that is the point of homeostasis). However, there are some fairly universal features and very important differences between the intra- and extra- cellular environments. Text books are likely to have lists that differ in detail or emphasis to what follows.... BUT the main points will be the same. All lists are equally "correct"

ExtracellularIntracellular
 Mg2+ 25 mM
Na+ 144 mMNa+ 10 mM
K+ 5 mMK+ 140 mM
Ca2+ 1 mMCa2+ 0.0001 mM
Cl- 110 mMCl- 10 mM
HCO3- 25mMHCO3- 10mM
HPO4- 7mMprotein- 65mM
SO4- 8mMPO4- 90mM

Not So different after all

Having made a big fuss about how different channels and primary and secondary active transport processes are one from another, at least from a functional physiological perspective, it is a bit weird to think that, structurally at least, the biggest difference between channels and all active transporters is how many "gates" they have. Channels have one and active transporters have two.

And finally...........

IMPORTANT IMPORTANT IMPORTANT

There are not many things in the first couple of weeks of the course that MUST stay with you for your entire Medical/Dental career, but knowing the difference between Na+ and K+ content the intra- and extra- cellular solutions is one of them.

You will find both Sodium (Na+) rich saline and potassium chloride (KCl) solutions on wards and clinics. The solutions themselves are indistinguishable.

If you mistakenly attach a KCl solution to a drip instead of a Na+ rich saline then YOUR PATIENT WILL DIE

It happens.

Make sure it never happens to you