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startled response to a nightmare or excessive exercise can suddenly kill some children who have long QT syndrome, a rare inherited disorder that impairs the heart's ability to conduct electricity.

Investigators have known since the mid-1990s that mutations in a slowly activating heart potassium channel, called KCNQ1-KCNE1, have been linked to many cases of the disorder, which shows up as a prolonged QT interval on an EKG.

But no one understood on a molecular level why the mutated channel would not work properly in response to activation of the sympathetic nervous system, which normally responds to stressful "fight or flight" situations.

Now, the laboratories of Dr. Robert Kass, chairman of pharmacology and Hosack Professor of Pharmacology (in the Center for Neurobiology and Behavior), and Dr. Andrew Marks, professor of medicine and pharmacology, Wu Professor of Molecular Cardiology, and director of the Center for Molecular Cardiology, have discovered the ensemble of proteins, coordinated by a protein called yotiao, that regulates this potassium channel, bringing researchers closer to understanding what initiates and causes fatal arrhythmias in the most prevalent variant of long QT syndrome. Their research findings, which also have implications for more common arrhythmias, were published in the Jan. 18 issue of Science.

Synchronized electrical activity is needed for proper cardiac function. Normally, the heart's pacemaker sends an electrical signal that is transmitted to each heart cell to initiate cellular contraction and, in turn, the heart beat. In the process, the signal changes the voltage gradient across the cellular membrane of the cells. To prepare the cell for the next signal, the cell's "resting" electrical potential must be restored, in part, by opening the potassium channels and allowing positively charged potassium ions to leave the cell. But in long QT patients, mutations in the channel limit its effectiveness to conduct "repolarizing" potassium current and delay the process of restoring cellular electrical properties for the next beat. When the heart is stimulated excessively by fright or exercise, the mutant channels do not allow adequate potassium efflux, resulting in electrical instability that puts patients at risk for sudden death from arrhythmias.

Before the Columbia study, other researchers had searched for molecules regulating the potassium channel. However, expression of the two subunits of the channel in cell lines could not reconstitute the channel's regulation by the sympathetic nervous system.

Dr. Kass and colleagues decided to employ a previously constructed fusion protein consisting of the two channel subunits expressed as a single protein to generate a transgenic mouse expressing a human form of the channel. The researchers then determined if the human channel responded to the sympathetic nervous system in the hearts and in cells isolated from these hearts. First, EKG recordings were recorded from transgenic animals treated with isoproterenol, a potent stimulator of beta adrenergic receptors, and the EKGs indicated that the channel was responding to this stimulation in the intact heart. Then, Junko Kurokawa, a postdoctoral fellow in Dr. Kass' laboratory, isolated single cells from ventricles of the transgenic mouse hearts and used high-resolution electrical measurements with single recording "patch clamp" electrodes, to measure ion currents through the expressed fusion protein channels. When stimulated by a chemical signal of the nervous system, the transgenic channel responded in a physiologically appropriate fashion. These experiments gave the researchers hope that they might be able to isolate regulatory molecules associated with the channel.

Meanwhile, studies in Dr. Marks' laboratory had shown a calcium channel in the heart needed for the heart's pumping action also was regulated by the nervous system. The group found the calcium channel required a complex of proteins (including a kinase that added a phosphate group to activate the channel, a phosphatase that removed the phosphate to deactivate the channel, and targeting proteins that brought the kinase and phosphatase to the channel) in order for the signals from the nervous system to activate the channel. On the basis of this discovery, Dr. Marks suggested that Dr. Kass add a phosphatase inhibitor to his preparations. The result: Dr. Kurokawa recorded enhanced potassium channel activity in the presence of a continually attached phosphate group.

The work in Dr. Marks' laboratory also led Dr. Steven Marx, assistant professor of medicine in the Center for Molecular Cardiology, to discover the exact structure in the calcium channel that allowed the channel and its regulators (the kinase, phosphatase, and a targeting protein that directed them to the channel) to be "zipped" together. When the zipper that brought the complex of proteins together was disrupted, the signals from the nervous system to the channel did not occur.

Dr. Marx, who works in the laboratory of Dr. Marks, noted that the potassium channel also contained a zipper structure and reasoned potassium channel regulation might be similar to that of the calcium channel. Investigators in Dr. Marks' laboratory used specific antibodies against yotiao (a targeting protein that acted in the calcium channel); PKA, a kinase; and PP1, a phosphatase, and showed these three proteins bound to the transgenic potassium channel and were present in human heart cells. How did the proteins regulate the opening and closing of the potassium channel? In response to signals from the nervous system, yotiao targets the kinase PKA to activate the channel by adding a phosphate and the phosphatase PP1 to deactivate it by removing the phosphate.

An important clue as to how a long QT mutation adversely affects the potassium channel came from the observation by Drs. Kass and Marks that the most common mutation of patients with long QT syndrome in Finland occurs in the region of the potassium channel that contained the channel's zipper, where yotiao and the channel stick together. When the researchers introduced this mutation into the channel and put the channel into cells, yotiao couldn't bind to the channel, and the signals from the nervous system couldn't activate the channel. Of all long QT patients, people with this common mutation have the highest risk of sudden death when startled or when they exercise.

The inherited form of long QT syndrome is rare, affecting 1 in 5,000 people. But the researchers think the protein complex may be involved in common heart arrhythmias. Neuroscientists might find their discovery relevant, too, as yotiao also is in brain cells. "Our goal is to take information from a rare disease and apply it to more prevalent diseases," Dr. Kass says. "A long-term goal is to develop novel molecular approaches to regulate this channel in a manner that may prove to be therapeutic for other cardiac arrhythmias that underlie sudden cardiac death in heart failure and some episodes of ventricular tachycardia."

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