Ion Channels and Excitable Cells

How does the heart beat out its regular rhythm for decades? How does a muscle cell contract, allowing us to move and breathe? How do our eyes process the light and color before them? In each case, specialized, excitable cells perform these activities by changing their electrical properties. These changes are regulated by the variety of ion channels found in the plasma membrane. What are ion channels, what kinds of ion channels are found in cells, how do they work, and how do they change the electrical properties of cells? These questions fascinate scientists and have been the subject of much research. Although the basics of how a cell becomes excitable have been understood for more than half a century, crucial details of how ion channels work are just now being discovered.

From 1939 to 1952, Alan Hodgkin and Andrew Huxley published a series of seminal papers that successfully described how the flux of ions across membranes is responsible for the generation of the action potential: an action potential is the transient, rapid rise and fall of the membrane voltage (Hodgkin & Huxley 1952a-f, Hodgkin et al. 1952). In general, action potentials occur in all excitable cells. The main question they sought to address was, how does an electrical signal propagate in a cell from one location to another? To answer this question they used the squid giant axon, since it offered several experimental advantages. It permitted them 1. to directly manipulate ionic concentrations — this kind of experiment had been performed earlier on isolated heart by Sidney Ringer — and 2. to use a new experimental technique, invented by Kenneth Cole, called the voltage-clamp to directly measure membrane currents. From their experiments, Hodgkin and Huxley were able to show that differences in ion concentrations exist between the inside and outside of cells, that the properties of the action potential depend on these ionic concentrations, and that transient changes in the permeability of the cell membrane to these ions accounts for the propagation of the action potential along the squid giant axon. Central to their theory was the construction of a mathematical model that proposed the existence of several ‘conductances' across the membrane. These conductances had specific properties, including selectivity for one ion over another, distinct kinetics. Although today we recognize these ‘conductances' as ion channels, at the time they were construed simply as mathematical terms in a series of equations used to describe the experimental data. For their work, Hodgkin and Huxley were awarded the Nobel prize in physiology and medicine in 1963. To this day their work stands as one of the best examples of how scientists can use mathematics to provide insights into complicated biological systems.

The next major advance followed from the recognition that ion channels are constructed from large proteins that reside in the membranes of cells (Figure 1). Using newly developed methods for molecular cloning, and in agreement with the biophysical studies of Hodgkin and Huxley, scientists described the amino-acid sequences for a variety of ion selective channels, including Na+, K+, Ca2+, and Cl- ion selective channels. However, Hodgkin and Huxley would never have been able to predict the diversity of channels that the new science of molecular genetics ultimately uncovered. Today, more than 100 genes for ion channels have been cloned in humans. Furthermore, we now recognize that there is tremendous diversity among ion channels such that they can be opened by different stimuli including voltage, temperature, pH, stretch, and ligands (Hillie 2001). These diverse channels have distinct biophysical properties that result in channels that open and close quickly, channels that open quickly but close only over long periods of time, channels that open slowly and remain open, channels that only permit ions to flow in one direction, and channels that remain open all the time! It is also important to emphasize that scientists now know that a cell can express many different types of ion channels, and that, together, this diversity of ion channels allows cells to finely tune their responses to different stimuli. However ion channels are not freely floating through the cell membrane like an iceberg in a sea of lipids. Instead, they are almost always anchored in specific locations within a cell, where they can perform a designated function (Lai & Jan 2006). For example, neurons can have a tremendously complex morphology, with long dendrites to receive synaptic input, and long axons to transmit signals from the brain to peripheral targets. In order for action potentials to traverse these very long distances in mammals, voltage-gated sodium (Na+) channels must be clustered at regularly spaced intervals along the axon. The mechanisms neurons use to regulate the kinds and locations of ion channels in a specific cell, and the contribution of the ion channel in its unique compartment to cellular physiology, are just now being discovered.

How does a channel selectively permit one ionic species to cross the membrane while excluding another? For example, how do potassium (K+) channels permit the flux of K+ ions while excluding Na+? Since Na+ ions are much smaller than K+ ions why do they not cross the cell membrane through the K+ channel? Ultimately, answers to these questions require an intimate knowledge of the structure of ion channels. The expectation is that, by deciphering the structure of a channel, it is possible to discover how the channel works. Unfortunately (from the experimenter's perspective), ion channels are large polypeptides with many hydrophobic membrane-spanning domains. This means it is experimentally very difficult to work with ion channels. For many years, physiologists depended on toxins and mutations in channels to probe their structure-function relationships. Because of this, our knowledge about the structure of ion channels was always indirect, and limited to channels for which toxins and DNA sequences were available.

However, in 1998, Rod Mackinnon and colleagues succeeded in what many people thought was an impossible experiment: they solved the X-ray crystal structure of a K+ channel (Doyle et al. 1998; Figure 2). They used a K+ channel from the bacterium Streptomyces lividans (named KcsA) since it offered several experimental advantages for X-ray crystallography (smaller size, fewer hydrophobic domains, and it could be expressed in bacteria in large quantities). Furthermore, KcsA appeared to conduct K+ ions just like mammalian K+ channels, and it could even be blocked by some of the same toxins used to study mammalian channels. There were many important discoveries that came from this work, but with respect to selectivity, Mackinnon and colleagues made an important and fundamental discovery about how the K+ channel structure is arranged to select K+ ions over others, specifically Na+. The channel does this by permitting carbonyl oxygens — carbon molecules with a double bond to oxygen — on the polypeptide chain to strip off the water molecules surrounding the K+ ions. These oxygens function much like a "cage" to confine the K+ ions (Figure 3). However, this structure cannot strip away the water molecules surrounding Na+ ions. Therefore, the effective radius of the Na+ ion — which includes the water molecules — is much larger than that for K+, which effectively excludes Na+ from the channel. This important work with the Kcsa channel was followed by others, as it also revealed the mechanism of high rate of ion flux through the channel. However, it did not reveal the mechanism of voltage-sensitive gating, and this question remains controversial. For his important work on the structure of ion channels, Rod Mackinnon shared the Nobel Prize in Chemistry in 2003. Since this first crystal structure of an ion channel, the structures of a few other channels have also been solved. Each has revealed different mechanisms permitting the flux of ions across membranes. Given the tremendous diversity of channels, and their importance in normal cellular function, the quest to solve more ion channel structures remains one of the most exciting and active areas of current research.

With the advances in molecular genetics described above, analyses of how defects in ion channel genes are associated with many human diseases has become possible. Indeed, epilepsies and cardiac dysfunction are just two examples of many illnesses stemming from mutations in ion channels. These are collectively referred to as channelopathies. For example, mutations in heart Na+ and K+ channels can lead to a cardiac channelopathy called Long QT syndrome (Ackerman 2004). People with these mutations are particularly susceptible to cardiac arrhythmias (Figure 4). With ever increasing ability to sequence whole genomes and perform genetic sequencing of patients, physicians and scientists now work together to discover how ion channel mutations lead to disease. Often, based on mutations identified in patients, scientists generate models that can recapitulate the effects of the mutation in a controlled environment. These models include genetically modified mice, cell culture models, and even tissues and cells collected from the patients themselves.

Ion channels are responsible for the transmembrane flux of ions that lead to the generation of action potentials. There is a stunning array of different types of channels that can be activated by different stimuli. Biophysical studies have begun to reveal the fundamental mechanisms responsible for the selectivity of a channel for one ion over another. And, both physicians and scientists are learning much about the role of ion channels in normal physiology from the discovery of human mutations. Nevertheless, the functions of many ion channels remain unknown, and their structure-function relationships are still undefined. Despite being very small structures, ion channels have large functions; they control the beating of a heart, the perception of sound or sight, or storage of a memory. Ultimately, these biological processes depend not on a single ion channel, but on all the ion channels in a cell and tissue network functioning in a coordinated manner. How this happens will continue to capture our imagination and attention for decades to come.