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A structural and mechanistic study of a voltage-dependent potassium ion channel--a membrane pore that shepherds K+ ions in and out of cells during nerve impulses and muscle contractions--published last month shows how this kind of channel opens and closes in response to changes in the distribution of charge inside and outside the cell [Nature, 423, 33 and 42 (2003)]. SHOCKER In MacKinnon's structure of a voltage-gated potassium channel, four subunits (blue) surround a central cavity that conducts K+ (red sphere). Voltage sensor paddles (light blue) studded with positively charged ions (red side chains) protrude into the lipid membrane. NATURE ©2003 The latest in a series of landmark achievements in the ion channel field by biophysicist, crystallographer, and Howard Hughes Medical Institute investigator Roderick MacKinnon of Rockefeller University, the study suggests that the widely accepted model of how voltage controls the opening and closing of voltage-dependent ion channels is wrong. Electrical impulses in nerve cells underlie processes as diverse as sensation, thought, and emotion. These electrical impulses are driven by action potentials, rapidly propagated changes in the balance of charge inside and outside nerve cells' membranes. During action potentials, sodium rushes into the nerve cells, making the inside more positive. Voltage-gated potassium channels sense such changes in charge distribution and open in response, allowing potassium to flow out of the cell. This discharge of potassium permits the nerve-cell membrane to return to its resting state and prepare for the next impulse. Because of sequence and functional similarities, voltage-gated K+ channels were expected to contain a central pore similar to that seen in the structure of a simple, voltage-independent K+ channel that MacKinnon's lab put forward a few years ago [Science, 280, 69 (1998)]. This archetypal channel revealed the molecular structure of the so-called selectivity filter that allows K+, but not other ions, to pass. It also provided a picture of the "gate"--the portion of the channel that moves to allow ions to access the selectivity filter. Not surprisingly, the selectivity filter and the gate seen in MacKinnon's structure of the voltage-gated K+ channel are nearly identical to that of its simpler cousin. Scientists have had fewer clues to go on to create a picture of the parts of the channel shown to sense voltage. From a variety of lines of biochemical evidence, everyone believed that the channel's voltage sensors were -helices "packed snugly among the other helices of the protein," notes Fred J. Sigworth, professor of cellular and molecular physiology at Yale University School of Medicine, in a Nature commentary that accompanies the two papers. CONVENTIONAL WISDOM in the ion channel community held that these a-helices--largely hydrophobic polypeptides studded with positively charged arginines--would slide or rotate through an aqueous protein pathway in response to a change in voltage across the membrane. Their concerted movement would in turn control the opening and closing of the channel. "This model has made it into the textbooks," Sigworth writes. "But the results of MacKinnon and colleagues show that it is almost certainly wrong." The molecular picture provided by MacKinnon, postdoc Youxing Jiang (now an assistant professor of physiology at the University of Texas Southwestern Medical Center), and their coworkers shows that the voltage sensors are actually paddle-shaped. Completely unexpectedly, these helix-turn-helix paddles protrude from the central ion-conducting pore like wings on a doughnut. This radical revision has ruffled some feathers in the ion channel field. Some, suspecting that the antibody fragments that MacKinnon's team used to hold the floppy voltage sensors in place in the crystals had dragged or distorted the paddles into an unnatural position or conformation, view the structure with suspicion. Others question whether the particular voltage-gated K+ channel that MacKinnon's team crystallized--a remarkably heat-stable one isolated from an archaebacterium found in thermal vents off the coast of Japan--bears any resemblance to those found in mammalian nerve cells. "We were shocked by the structure," MacKinnon admits. "The voltage sensor looked nothing like we had imagined." His team quickly set about designing a series of biochemical experiments to test whether the crystal structure had any basis in reality. In light of the large body of functional data that's been amassed on these channels, MacKinnon and his team realized that the antibody fragments were causing some distortions in the structure, and they set out to determine how the paddles might move in response to voltage changes. This required antibody-free experiments with the membrane-bound, full-length channel in which a tethered biotin molecule was attached to various positions on the voltage sensor. Once attached at certain positions, the tethered biotin molecule is dragged from the inside to the outside of the membrane when the channel opens in response to a voltage change. The bulky tethered biotin molecule can't be dragged through the protein environment, as would be required by the conventional model, MacKinnon argues. Instead, he says, it has to be moving through the membrane itself. Furthermore, the paddles move 15–20Å--far more than the conventional model predicts. TEAMWORK MacKinnon (top row, far left) credits his continued success to the hard work and determination of his coworkers, who in this case included (front row, from left) staff scientists Alice Lee and Jiayun Chen and (top row, from left) graduate student Vanessa Ruta and postdoc Youxing Jiang. PHOTO BY LYNN LOVE/ROCKEFELLER UNIVERSITY -------------------------------------------------------------------------------- WITH THESE RESULTS in mind, MacKinnon's team suggests that the voltage sensor paddles act as hydrophobic cations attached to levers. In resting nerve cells, these positively charged levers remain tucked in next to the closed pore, near the intracellular (more negatively charged) side of the membrane. As nerve impulses are propagated, nerve cells become more positive inside and more negative outside--forcing the positively charged levers to flip up toward the extracellular side. This movement tugs the channel open, and K+ ions flow out of the cell, allowing the nerve cell to return to its resting state. Amino acid sequence conservation indicates that the voltage-sensor structure will be similar in potassium, sodium, and calcium channels. In fact, other voltage-gated channels may use a similar voltage-sensing strategy, MacKinnon notes. California Institute of Technology crystallographer Douglas C. Rees recently showed that an unrelated channel--which lets ions through indiscriminately in response to voltage and mechanical force--has similar arginine-studded hydrophobic paddles flanking its central pore, pointing into the lipid membrane [Science, 298, 1582 (2002)]. Still, there are a large number of experimental observations in eukaryotic channels that cannot be reconciled with MacKinnon's model, according to neuroscience professor Francisco Bezanilla of the University of California, Los Angeles, and physiology professor Diane M. Papazian of UCLA's School of Medicine. MacKinnon admits that there are some pieces of data--including work from his own lab--that are hard to explain with his model. But, he adds, the model is "very consistent" with much of the other data gathered on voltage-gated channels. To sort out the details, his lab is working to obtain crystallographic snapshots of the channel in a variety of conformations by using antibody fragments directed to different portions of the channel. "As has become the norm, MacKinnon's intriguing work has taken the field by storm," says Eduardo Perozo, associate professor of molecular physiology and biological physics at the University of Virginia Health Sciences Center. Perozo adds that quite a bit more experimental work is still needed to reach a consensus |
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