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NEUROBIOLOGY Ion Channels Hold Clues to Drug ActionMelding molecular genetics and biophysics, Gary Yellen probes the workings of ion channels—proteins that govern electrical activity in the nervous system, heart, and muscle. His lab's latest discovery is that a potassium channel found in neurons probably differs in a critical way from a bacterial potassium channel whose crystal structure has until now guided most scientists in the field. Reported in the Jan. 20 Nature, "the paper represents a fundamental change in our understanding of one of the key switching structures in nerve cells," says Yellen, HMS professor of neurobiology.
Instructor Miguel Holmgren, postdoc Donato del Camino, and Gary Yellen (l to r), as well as former postdoc Yi Liu (not shown), combined site-directed mutagenesis and chemistry with electrophysiological recordings to figure out precisely where blockers bind to potassium channels. Similar blockers serve as local anesthetics. The study of what ion channels look like and how they open and close continues a classic line of basic neuroscience. It began with the initial description, early this century, of the sodium, potassium, and chloride currents that generate an action potential, and had another high point in the study of the channel proteins and blocking agents that garnered last year's Albert Lasker Award for basic medical research. But these days, the field is taking on additional urgency as clinical neurologists and gene hunters have begun putting diseases ranging from heart arrhythmia to deafness on a rapidly growing list of "channelopathies." "In the past decade, the idea that human disease can be caused by inherited mutations in ion channels has become a hot topic. Until then, people thought these would always be fatal because channels are so fundamentally important," says Stephen Cannon, HMS associate professor of neurobiology (see sidebar). Research such as Yellen's can help explain how such mutations alter the function of the affected pore. It can also yield insight into how drugs treating these conditions work and provide clues for designing more specific ones. Where's the Gate?To appreciate the importance of ion channels, consider that a cell typically expends about 30 percent of its energy to maintain the uneven distribution of ions on either side of its membrane, which gives the membrane its electric potential. Ion channels form pores spanning the membrane and tap into this potential every time they open, like a switch releasing a battery's stored energy. Once open, roughly 10 million ions per second flow through each pore.
The inner part of a bacterial potassium channel roughly resembles an inverted teepee (left). To stop the flow of ions out of the channel, its poles probably rearrange at their crossing point, or smokehole, pinching off the lumen. Researchers in the Yellen lab realized that the neuronal K+ channel must look different when they probed its inside with blockers. Unexpectedly, large blockers (ball peptide) penetrated up to the smokehole. This suggests that the poles in neuronal potassium channels do not form a cone-shaped inner opening but instead bend outward, creating a broad vestibule at the channel's interior entrance (right). Cysteine and histidine side chains (red and black circles) that can form bonds to lock the channel open, give further evidence for the bent structure. Yellen studies a potassium (K+) channel from fruit flies that is closely related to mammalian K+ channels. At their most basic, these channels restore the membrane potential to its former strength after action potentials have reduced it. But they also govern neurons' rhythmic firing patterns and translate the intensity of a sensory stimulus—say, a bright vs. a dim light—into properly coded salvos of action potentials. For years, Yellen has tried to find where in the channel its moving parts, or gates, lie and how they open and close in response to changes in the membrane potential. This is not a straightforward task in a large protein that comprises four subunits, each of which crisscrosses the membrane six times. Scientists already know that ion channels are complex, able to open and close in different ways. They are not like a concrete tunnel with doors on either end; rather, they can constrict and relax at several stretches along their length. Some also feature a round peptide tethered to the interior end of the channel that can swing up and plug the channel from the inside. A Channel Shapes UpIn previous work searching for such a gate, Yellen's team replaced amino acids facing the channel's inner pore with cysteine, which allowed the scientists to set up a chemical reaction along the channel's lumen that they could monitor through its effect on the current flow. Then they asked: Which of these cysteines can the reagent no longer reach when the channel closes? Given that the reagent enters from the channel's interior side, all sites above the gate should become inaccessible once the gate closes. This 1997 study suggested a gate that was given a physical shape when Roderick MacKinnon published, in 1998, the first, and still only, crystal structure of a K+ channel. One of the Lasker Prize recipients, MacKinnon left HMS in 1996 for Rockefeller University in New York.This structure, of a bacterial K+ channel, depicts an inverted teepee, whose poles cross near the intracellular end of the membrane to create a "smokehole" that appears to constitute the gate (see image). The structure immediately became the working model for a field starved for direct structural information. Yellen's present paper, however, shows where the similarities end. Yellen and his colleagues reached their conclusion serendipitously when they continued their previous work with the cysteine-based reporter reaction. This time they asked precisely where blockers known to inhibit this channel actually bind, by checking which of the cysteine reactions disappeared because a blocker was sitting on that section of the channel. The scientists used blockers ranging from small to large, reasoning that the narrow opening below the smokehole should preclude the larger blockers from penetrating the channel (at left in image). To their surprise, they noticed that even the largest blocker—the channel's own ball peptide—slid up into the channel almost all the way to the smokehole. This was odd, so they looked for other clues to the neuronal K+ channel's structure. They found two: first, the channel's DNA sequence suggested a likely kink in the teepee poles right at the smokehole. Second, another previous finding made more sense when explained by a bent structure. Taken together, all this suggested that the teepee's poles bend sharply, pointing outward and lying almost flat against the inside of the membrane (right channel in image). To be sure, the crystal structure remains highly relevant to the field, Yellen adds. "Overall, it is still closely related. But there is a critical difference right where the action is." One payoff of this work lies in an improved understanding of channel blockers, such as epilepsy and heart arrhythmia medications. Channel therapeutic drugs are unusual in that they act on some states of the channel more strongly than on others, whereas unconditional channel blockers are toxins—a fact exploited by poisonous snakes, scorpions, and anemones. Some epilepsy medications, for example, dampen excess channel opening leading to seizures—but leave normal activity alone. Channelopathies—En Vogue in NeurologyScientists began to realize 10 years ago that faulty ion channels might cause inherited diseases. By now, they blame about 30 conditions, including some forms of epilepsy, migraine headaches, and kidney disease, as well as muscle disorders, on subtle changes in channel function. The first example of a channelopathy emerged in 1990, when researchers at Massachusetts General Hospital established a genetic link for hyperkalemic periodic paralysis. People with this rare disease suffer episodic attacks of paralysis that can render them unable to raise a limb for minutes, hours, even days. A year later, Stephen Cannon, HMS associate professor of neurobiology and a neurologist at MGH, reported the first functional defect in a sodium channel, and shortly thereafter, researchers at MGH and elsewhere pinpointed the first mutation. Since then, mutations in sodium, chloride, and calcium channels have proved responsible for six different muscle diseases. Some of these stand out in that scientists, including Cannon, have traced their complete story. They know the mutations in the channel gene, how the mutated protein behaves, and how that behavior changes the muscle's excitability, which explains the patient's symptoms. Less well understood is epilepsy. Affecting up to 2 percent of the population in at least 1,000 different forms, this large group of seizure disorders poses a much more complex problem. Yet several types of epilepsy are now known to be due to altered channels, says Elizabeth Thiele, an epileptologist and instructor in neurology at Children's Hospital. "All the epilepsy genes found so far turned out to code for ion channels, but only in the last couple of years have people appreciated that so many forms of epilepsy are genetic," she says. An intense effort is under way to map epilepsy genes, she adds, but the field is still far away from its goal of linking certain epilepsies to particular channel types and then designing more specific drugs that lack the systemic side effects of current treatments. |
