Basic Medical Research Award
Clay Armstrong, Bertil Hille and Roderick MacKinnon
For elucidating the functional and structural architecture of ion channel proteins, which govern the electrical potential of membranes throughout nature, thereby generating nerve impulses and controlling muscle contraction, cardiac rhythm, and hormone secretion.
Biologically speaking, human beings are frequently described in a number of ways. We are flesh and bone. We are comprised of essential life-giving organs neatly arranged beneath a sheath of skinitself an organ of astounding complexity. We are made up of cells, genes, DNA.
It is less common to think of humans as electrical beings, but the description is equally apt. Like any good machine, humans are controlled by electricitybodies made up of a vast network of interactive electrical components that surely surpass in intricacy those of any supercomputer.
This year's winners of the Albert Lasker Award in Basic Medical Research bring to their work a unique appreciation of biology and a keen understanding of electricity.
Clay Armstrong is honored for his work in "cell membrane excitability" and the elucidation of "ion channel gating kinetics." Armstrong himself puts it more simply: "My research involves electricity. Electrical signals are used throughout the nervous system and activate muscle cells. Essentially, we studied the electrical system that underlies all thinking and movement."
As a medical student at Washington University in the late 1950s, Armstrong was captivated by lectures on the physiology of nerve impulses and by the work of A.L. Hodgkin and A.F. Huxley, who did pioneering studies of the squid, an elegant model for analysis of the passage of nerve impulses through cell membranes. Hodgkin and Huxley showed that the action of nerve cells is dependent on electrical conductance changes in the cell membrane. But the physical structures underlying these changes remained mysterious.
That is where Armstrong took the field to its next level, extending the Hodgkin-Huxley hypothesis as he himself adopted the squid as his experimental model for biophysical studies of the electrical system. Throughout, he always kept in mind the potential application to medicine of lessons learned from squid. "If all nervous system cells operate electrically, then determining the electrical mechanism has to be important," he thought. Ion channels were the first step.
In order to understand the significance of the work of Armstrong and his co-winners, Hille and MacKinnon, it is important to understand ion channels as the basic component of the body's electrical system. It is a concept that many people find difficult to grasp. Again, Armstrong makes it simple. "Ion channels are little holes in the membrane of all cells. The channels open and close to either permit or block certain ions from crossing the membrane. Sodium, potassium, calcium, and chloride channels are among the most important molecules in the electrical signaling system."
A signal is generated when sodium or calcium ions move into the cell, while chloride and potassium ions inhibit signal generation. For example, by blocking calcium's passage through ion channels, inappropriate signals are prevented and the heart beats as it should. Today, the idea that some cardiac drugs are calcium channel-blockers is commonplace, but in the 1970s, Armstrong, and his co-winner Bertil Hille, were among the few scientists who believed that ion channels even existed.
Between 1964 and 1971, Armstrong developed evidence for gates that control the movement of ions into or out of a cell. He envisioned the ion channel as a long, thin pore, flanked by slightly wider "vestibules." The narrow part of the pore determined its ion selectivity. Ions coming from one end could dislodge blockers that had entered the channel from the other end, providing clear evidence for the channel idea.
Hille describes Armstrong's accomplishment this way: "He proved for the first time that a drug may block an ion channel by physically plugging the pore." In some cases, an ion blocks the pore from the inside, as if "swept into the channel by the electrical field." Alternatively, external "in-rushing" potassium ions can clear the "plug," or block, from the channel in the membrane. In all cases, a drug can neither get into nor out of a cell unless the potassium channel gates are open. The study of potassium channel-blockers gave a surprisingly detailed picture of channel structure.
Throughout the 1970s, Armstrong and his team proposed what is called the "ball and chain" model of channel inactivation, demonstrating that the activation and inactivation of ion channels involves two separate physical structures. An "activation" gate covers the inner end of the channel. When this gate swings open after a change in electrical voltage, the channel conducts transiently and then a tethered peptide "ball" swings into place to block or inactivate the channel. The ball is held in place by a chain that can be severed by enzymes, leaving the activation gate unaltered.
Armstrong's simple mechanical picture of a complex electrical system drew considerable skepticism in the beginning, but now is generally accepted.
Finally, Armstrong suggested a powerful explanation of the electrical or voltage dependent changes that precede the actual opening of an ion channel when he showed that the channel's activation gate itself is comprised of one positively charged and one negatively charged helixin short, a zipper.
Now Armstrong is focusing his energy on the idea that proteins in solution are key to understanding the phenomenon of ion channel gates at an even more basic level. And once again his notions are being met with doubt by some of his colleagues.
But as Hille says, "Clay has intuitions that seem to go well beyond the experiments and that much later seem to be right on."
Bertil Hille, who shared Clay Armstrong's belief in ion channels long before the proof was in, says they envisioned "holes in membranes" in the early 1960s and were convinced that the "holes have gates that open and close. But we were working on a black box, with none of the equipment that exists today. We believed that ions have to go through the right size hole to get into a cell, so the holes or channels must come in different sizes. And we wanted to know whether the gates that control ion flow are on the inside or outside of channels."
Hille's interest in the field took hold while he was a graduate student at The Rockefeller University, from which he received his Ph.D. in 1967. It was Hille, working with nerve axons from frogs, who showed that channels are independent physical entities in the membrane, each site generating electrical signals that make it possible for cells to talk to one another. Selective block by several neurotoxins proved that there are discrete and separate channels for sodium ions and for potassium ions. Prior to Hille's discoveries, many scientists assumed that ions could flow across a membrane at any point.
In addition to identifying specific channels in cell membranes, Hille was fascinated to know how a molecular pore could recognize the difference between the tiny sodium and potassium ions. He demonstrated that the channels are sized to accept one kind of ion or another. Their pores have the capacity to act as a molecular sieve. He established that the pores contain water molecules, which also contribute to the ion selectivity.
Hille's description of ion flow in sodium channels explained how only one sodium ion can penetrate or permeate a channel at a time, shedding water molecules as it passes through a series of energy states before getting to the inside of a cell. In contrast, his description of potassium channels explained many of their known properties by assuming that there are several potassium ions in that pore at once.
In 1977, Hille, always keeping an eye on the medical implications of his research, described the molecular interaction of local anesthetics acting on sodium channels, thereby laying the foundation for understanding the mechanisms of anesthesia. "We discovered that many agents, including local anesthetics, are channel-blocking agents. They wait until the door is open on the inside of a sodium channel, then enter and sit in the pore so no sodium can enter the cell. Therefore, no electrical signal gets to the brain and pain is blocked. This picture borrows much from Clay Armstrong's potassium channel work of ten years earlier."
Hille's study of these blocking agents also contributed to pharmacological understanding of the action of arrhythmic drugs on the heart.
Now, Hille's research is directed to the role of ion channels in a variety of cell systems, particularly G-protein signaling and the control of neurotransmitters such as adrenaline, acetylcholine, serotonin, and dopamine. "We originally thought that only nerve cells had ion channels. Then we added muscle cells, and now we know that every cell has ion channels to make signals."
Hille notes that everything from sperm to white blood cells to endocrine glands needs ion channels. Furthermore, he adds, "the number of kinds of known channels has grown as well. A single excitable membrane may contain five to ten kinds and our genome codes for more than 100."
Hille is not only an original thinker; he is also a remarkable author and teacher. Virtually all of his admirers mention Ionic Channels in Excitable Membranes, a classic text first published in 1984. It is considered the scholarly bible of ion channels and is one of the more cited publications in the scientific literature. Hille currently is preparing the third edition.
Roderick MacKinnon is honored for his elucidation of the structure and function of potassium channels. His work provided the first molecular description of an ion selective channel.
MacKinnon's achievement is described in this way by a colleague: "In only a decade, Rod MacKinnon has taught us how a potassium ion channel is built and how it can unerringly pluck potassium ions from a sea of sodium ions and conduct them [through the channel] at a rate approaching the diffusional limit." And, through his work in crystallography, he uncovered "a structure of breathtaking beauty that reveals how evolution satisfied the apparently paradoxical requirements for high selectivity and high throughput. MacKinnon has forever changed the way we view all ion channels."
MacKinnon graduated from Tufts University School of Medicine in 1982, but four years later he abandoned plans to practice medicine in order to pursue postdoctoral studies at Brandeis University, in the laboratory of his undergraduate mentor, Christopher Miller. "My scientific career in effect began at the age of thirty," he says.
In Miller's laboratory, MacKinnon began working on biophysical aspects of channel function. "I focused on the protein selective for potassium ions, because it happened to be the target of our lab." In the past 13 years, MacKinnon has sought the answers to two compelling questions: What do potassium channels look like? And how do they work?
MacKinnon, well known for his penetrating analyses of ion channel function, adopted a formidable array of techniques to answer these questions. Using electrical measurements, MacKinnon deduced that a scorpion toxin blocks a potassium ion channel's pathway. He then exploited the toxin to analyze the subunit structure, moving gates, and ion conduction pathway of potassium channels.
MacKinnon and his biophysics team combined electrophysiology and molecular biology to identify the potassium channel pore loop. Through a series of elegant experiments, they showed that the pore loop defines the essence of a potassium channel by forming its selectivity filter, conferring the ability to accept potassium and exclude sodium. The central feature of the pore loop is the "signature sequence," found in all potassium channels from animals to plants to bacteria.
Little more than three years ago, MacKinnon made the boldest of his career decisions. He chose to extend his conclusions based on electrophysiology and molecular biology by focusing most of his effort on obtaining the crystal structure of a potassium channelconsidered by many the Holy Grail of ion channel biophysics.
His research team at The Rockefeller University focused on the KcsA channel, a potassium channel with two membrane-spanning segments and the signature sequence. They crystallized the KcsA potassium channel and solved its structure at 3.2 Å resolution, sufficient to answer the classic questions raised in the Hodgkin-Huxley age of membrane biophysics: What is the physical nature of an ion channel? What are the determinants of ion conduction and selectivity?
The channel, as MacKinnon had deduced earlier, is a tetramer of symmetrically arranged sub-units. Four inner helices are arranged like the poles of an inverted teepee with the wide end near the outer side of the membrane. The pore loop amino acids form the selectivity filter in the teepee's wide end, where potassium ions on their journey across the membrane interact with backbone atoms from the signature sequence amino acids.
Most impressive was the stunning visualization of three ions within the poretwo in the selectivity filter and one in a cavity near the membrane center. "Years of pharmacology and ion permeability studies were suddenly understandable at a new and deeper level," says MacKinnon.
Although the potassium channel can be elegantly described in the language of molecular biology, MacKinnon (like Armstrong and Hille) has the gift to put things simply. "Biological systems have small components that enable them to produce electrical activity. They are not entirely unlike toasters or televisions that have resistors, capacitors, and various other little parts. The potassium channel is one of the basic components or pieces of hardware in the body that carries ions to create electrical activity." Of course, electrical activity is essential to life.
Armstrong and Hille were thrilled by MacKinnon's paper. "It was the very first time we actually saw an ion channel," says Hille. MacKinnon proved beyond doubt that many of the original hypotheses of Armstrong and Hille were right. Clay Armstrong calls MacKinnon's structure "a dream come true for biophysicists."
As is characteristic of great scientists, Armstrong, Hille, and MacKinnon are pursuing ion channel studies to achieve a more thorough comprehension of how the living molecules are built and how they work.