Understanding ion channels

Professor Peter Agre of Johns Hopkins University at a press conference after being named the joint winner of the Nobel Prize in Chemistry. - ROBERTO BOREA/AP

Two doctors share the Chemistry Prize for research on the transportation of uncharged water molecules and the charged ions of sodium and potassium salts in aqueous solution in and out of cells.

A HUMAN being has about one hundred thousand million cells in the body, and the cells - muscle cells, kidney cells and nerve cells - form an intricate system acting in a well-coordinated manner. Crucial to this function is the transportation of uncharged water molecules and charged ions of sodium (Na) and potassium (K) salts in aqueous solution in and out of cells. This year's Nobel Prize in Chemistry has been awarded jointly to two scientists whose work has led to the understanding, at molecular level, of how the cells achieve this selectively, while remaining impermeable to other atoms.

The 54-year-old Peter Agre, a medical doctor from Johns Hopkins University School of Medicine, United States, and now Professor of Biochemistry and Medicine at the same place, unravelled the structure and chemistry of water channels in cells. Roderick McKinnon (47 years), a medical doctor turned scientist and Professor of Molecular Neurobiology and Biophysics at The Rockefeller University in New York, U.S., on the other hand, found out the molecular chemistry of ion channels.

In some sense, the works that won this year's Nobel prizes in Medicine (for work related to Magnetic Resonance Imaging or MRI) and Chemistry are complementary to each other. While MRI is chiefly used to map the water content in cells and tissues, the work of this year's Chemistry laureates tells how water is transported through the cells and how signals from the cell propagate to enable the coordination of muscles and nerves. This is of utmost importance in the understanding of many diseases. A number of diseases can be attributed to the poor functioning of water and ion channels in the body. The kidneys recover water from primary urine. The failure of this absorption process can arise in diabetic conditions and MRI imaging can tell us the extent of the kidney dysfunction. The knowledge of the structure and function of molecular channels now enable proper therapeutic intervention.

As early as the mid-19th century it was known that the cells must contain specific channels for transporting water and salts. In the mid-1950s it was discovered that water could be transported rapidly in and out of cells through certain pores that admit water molecules only, and not ions. Subsequent studies indicated that there must be some kind of `selective filter' that prevents the (charged) ions from passing through while water molecules, which are chargeless, flow freely. A single water channel can transport over a billion water molecules per second! However, the molecular machinery that enables this selectivity had to wait till the arrival of Agre's work in 1992. In fact, the very idea of a water specific channel remained controversial until then. Like all other body functions, a specific protein was involved.

In the mid-1980s, Agre studied several membrane proteins from red blood cells. In 1988, he isolated a new protein of unknown function that he also found in the kidney. It was called CHIP28. After determining its terminal peptide sequence and the corresponding whole complementary DNA (cDNA) sequence, he realised that this must be the elusive water selective channel protein. Soon, Agre provided a conclusive demonstration of his hypothesis by doing a simple experiment. He compared the egg cells of the organism xenopus with and without the protein being expressed in them. The cells with the protein swelled rapidly by absorbing water by osmosis, while the cells that lacked the protein remained the same. The same phenomenon was observed with artificial soap bubble-like cells known as liposomes, which contain water inside and are also surrounded by water. In both cases, swelling was found to be inhibited by the addition of mercury ions, a treatment known to block water transport across the red cell membrane. He named the protein aquaporin, "water pore" and it was christened aquaporin 1 (AQP 1).

Aquaporin-like proteins have since been found in all organisms. In human beings alone, there are 11 different AQP-like proteins, many of which have been linked to various pathological conditions. Plants have even higher number of such proteins. For example, the model plant Arabidopsis thalima has as many as 35 different types.

The medical importance of AQP is perhaps most apparent in the functioning of the kidney. Kidney reabsorbs as much as 150-200 litres of water from primary urine every day through a series of mechanisms so that finally about one litre leaves the body as urine. This is made possible by AQP1 and AQP2. Primary urine passes through a winding tube where about 70 per cent of the water is reabsorbed by the action of AQP1. At the end of the tube, another 10 per cent is absorbed by the action of AQP2. Apart from this Na, K and chloride ions are also reabsorbed into the blood. Antidiuretic hormone (vasopressin) stimulates the transport of AQP2 to cell membranes in the tube walls and thus enhances the water absorption. People with a deficiency of this hormone are affected by nephrogenic diabetes insipidus, as well as with several conditions associated with water retention such as congestive heart failure resulting in a daily urine output of 10-15 litres.

BUT how does the water channel work? During 2000-01, together with other research groups, Agre reported the first high-resolution images of the 3D structure of AQP. With this, it was possible to map in detail how a water channel functions by selectively allowing only water molecules to pass and not other molecules or ions. The cell membrane, for instance, cannot allow the leakage of protons (or, more precisely, positively charged oxonium ions H3O+). This is crucial because the difference in proton concentration between the inside and outside of the cell is the basis of the cellular energy storage system. The protein AQP achieves this by an ingenious mechanism.

Water molecules pass through the narrow channel in a single file by orienting themselves in the local electric field formed by the protein molecules in the channel wall. The wall has a residual positive charge somewhere in the centre of the channel. Therefore, positively charged protons are repelled. This prevents proton leakage through the channel.

Thus, in the short span of 10 years, a complete molecular understanding of water channels has been possible, their physiological significance understood and their role in health and disease are now topics of intense research activity. Agre's unexpected discovery of aquaporins thus revolutionised the study of water transport and laid the basis for a new area of physiology and medicine.

As early as 1890, Wilhelm Ostwald (Chemistry Nobel laureate, 1909) suggested, on the basis of his experiments with colloidal membranes, that electrical currents in tissues are caused by ions moving across cell membranes. The idea that ions signal with salts also came to be generally accepted. It was further confirmed by work in the early 1900s that established that membrane potentials are electro-chemical in nature. In 1925, the existence of narrow ion channels was proposed.

In the early 1950s, British scientists Alan Hodgkin and Andrew Huxley, made a major breakthrough and showed that ion transport through nerve cell membrane produces a signal that is transmitted from nerve cell to nerve cell much like a relay race. Primarily positively charged sodium (Na+) and potassium (K+) ions are involved in the reactions. Their findings led to a detailed model for the ion channels, which opened and closed much like a gate by the action of membrane potentials. It was also demonstrated by them that K ions moved in a single file indicating that the selective transport of ions occurred through channel structures embedded in the membrane. They demonstrated all this using squid nerve extensions known as axons. Thus their work clarified the central concepts of ion transport - ion selectivity, channel gating and channel inactivation (or closing). They ushered in the new era of neurophysiology and were awarded Nobel Prize in Physiology and Medicine in 1963.

However, underlying molecular mechanisms remained totally unclear. During the 1970s, it was further shown that ion channels were equipped with some kind of `selective filter' to allow only certain types of ions. Interestingly, even though sodium ions are smaller than potassium ions, only the latter pass through the channels. It was speculated that perhaps oxygen atoms in the protein played a crucial role by behaving just like the water molecules with which the potassium ions surround themselves outside the cell in a stable configuration. To enter the channel, a potassium or sodium ion has to free itself from the water molecules.

But without a detailed understanding of the chemical structure of the channel this hypothesis could not be substantiated. What was needed was high resolution-images using the technique of X-ray crystallography. But determination of protein structures using this method is extremely difficult. Membrane proteins from plants and animals are, in fact, more complicated to work with than those from, say, bacteria. One possibility was to identify bacterial channel proteins that closely resembled human ion channels and determine their structures.

In 1998, Roderick MacKinnon - a medical doctor who gave up his profession and started doing research because he "just had to solve" the problem of ion channels - stunned the scientific community by determining the 3D structure of a protein that controlled the potassium ion channels, something many other researchers before him had failed to achieve. He determined the first high-resolution image of an ion channel from the bacterium Streptomyces lavidens. The ion filter could now be studied in detail.

The most tedious part, according to MacKinnon in this effort was the purification of proteins and obtaining sufficient quantities of good crystals. Membrane proteins are known to take notoriously long to crystallise and it can take many years. MacKinnon's painstaking research involved screening a large number of environmental conditions to identify the optimum set for crystallisation. His group managed to solve the problem in two years.

MacKinnon's study showed that since the distance between the potassium ion and the oxygen atoms in the channel is the same as the distance between potassium ions and oxygen in the water molecules surrounding the ion when it is outside, the same stable energy configuration is obtained and the potassium ions pass without hindrance and with no loss of energy. Potassium ions are equally happy in water and in the ion filter. For sodium ions, however, it is different. The distance within the protein filter is greater - because the sodium ion is smaller - than the corresponding distance in water solution and so they do not `fit' between the oxygen atoms of the filter. So the sodium ion prefers to stay in the stable energy configuration and does not enter the channel.

The closing and opening of the ion channels had also to be understood. MacKinnon showed that a gate at the bottom of the channel, achieved this, which opened and closed a molecular `sensor'. These molecular sensors are of different kinds that react to different kinds of external signals.

For example, changes in concentration of calcium ions, an electric voltage over the cell membrane and so on. By connecting different sensors to ion channels, a signalling network is set up in the nervous and muscular systems of the body that respond to different stimuli and a large number of signals to cause various muscular and nervous activities. Central to this understanding is MacKinnon's work on how the ion channels and their selective filters work.