Predicted structural model for the sodium channel C-terminal region, highlighting the EF-hand loop in yellow, with a blue calcium ion bound. The red amino acid (bottom center) is one that is mutated in long QT syndrome, which predisposes patients to life-threatening arrhythmias.
Vanderbilt team uncovers molecular clue to arrhythmias
Vanderbilt University investigators have tracked a question about heartbeats all the way from the clinic to the Center for Structural Biology. Their studies, reported this month in Nature Structural & Molecular Biology, reveal the molecular basis for certain arrhythmia-producing syndromes and suggest a target for new antiarrhythmic drugs.
Cardiac arrhythmias — irregularities in the heartbeat —are the leading cause of sudden death in the United States.
Certain individuals with inherited mutations in the cardiac sodium channel gene suffer from a condition called long QT syndrome that predisposes them to life-threatening arrhythmias. Jeffrey R. Balser, M.D., Ph.D., assistant vice chancellor for Research, and colleagues have been probing the function of sodium channels, trying to ferret out how mutations in the donut-like pores cause long QT syndrome and its resultant arrhythmias.
The investigators knew that a cluster of mutations in a specific region of the sodium channel — the C-terminal region — was associated with long QT syndrome. But how these mutations affected channel function has been an open question, until now.
Balser and graduate student Tammy L. Wingo approached colleagues in the Vanderbilt Center for Structural Biology for assistance, and a fruitful collaboration was born. The team, including center members Terry P. Lybrand, Ph.D., Walter J. Chazin, Ph.D., and graduate student Vikas N. Shah, has identified a structural motif called an EF-hand — known to bind calcium in other proteins — in the C-terminal region of the sodium channel.
The group further demonstrated that this domain in the sodium channel binds calcium, that calcium influences the activation state of the sodium channel protein, and that one of the mutations underlying a particular form of long QT syndrome disrupts calcium binding by the EF-hand domain.
“This work is a beautiful example of what the NIH roadmap calls interdisciplinary research,” said Chazin, director of the CSB and professor of Biochemistry and Physics. “It started in the clinic and reached all the way into physical biochemistry, and that’s a very great distance. Vanderbilt is an absolutely superb place for this sort of thing to happen.”
The project really took off when graduate student Tammy Wingo and Balser went to Lybrand, professor of Chemistry and Pharmacology, seeking to use computational biology — one of the methodologies of structural biology — to search the sodium channel’s C-terminal region for likenesses to other known proteins. “We wanted to start to understand what the C-terminus looks like and how mutations in that region might generate arrhythmia phenotypes,” Wingo said.
When the computational methods identified a putative EF-hand domain, Chazin came on board, bringing his 20-plus years of experience studying calcium-binding proteins and calcium signaling. After working with the structural biologists to create a model of the region, Wingo returned to the electrophysiological methods of the Balser lab, with assistance from Mark E. Anderson, M.D., Ph.D., to understand how calcium might affect sodium channel function. In parallel, Shah carried out the detailed biophysical analysis of the calcium-binding domain.
“We saw very clearly that calcium was affecting the inactivation gating process of the channel,” Wingo said. That process, she said, determines how many sodium channels are available to let sodium pass across the heart cell membrane. Sodium passage plays an important role in the electrical spark of each heartbeat.
The structural model of the region showed that one of the mutations that causes a particular form of long QT syndrome was in one of the key calcium-binding positions within the EF-hand, disrupting the ability of calcium to bind to the domain and influence sodium channel function.
The studies will guide antiarrhythmic drug discovery efforts, Chazin said. “By using these approaches to determine exactly what’s wrong, we know what kind of therapy we need to fix the problem. So in this case, we want a drug that will restore whatever it is that calcium binding does to the C-terminus of the channel, and we have logical leads for that based on what we know about other calcium-binding proteins.”
The challenge, Chazin added, is that cells are full of calcium-binding proteins, so finding a drug that affects only the sodium channel could be difficult.
The efforts may also impact treatments for patients in the aftermath of a heart attack. “One of the biggest killers of patients who survive their myocardial infarctions are ventricular arrhythmias in the one or two days following the MI,” said Shah, a student in the M.D./Ph.D. training program. The maintenance of sodium channel function in the face of elevated intracellular calcium seems to be crucial to avoiding these fatal arrhythmias, Shah said, suggesting that therapies directed at the EF-hand domain might also be useful for these situations.
The collaborative effort is a perfect example, Chazin says, of how the Center for Structural Biology is designed to operate. “The Center is a resource to learn, think about, and apply structural thinking to medical and biological problems. We’ve got a group of investigators who are all structural thinkers, and part of our mission is to demonstrate how powerful this approach can be. We’re highly motivated to collaborate and work with our colleagues to achieve these results.”
The Vanderbilt Center for Structural Biology (http://structbio. vanderbilt.edu/) is unique, Chazin added, in intermingling the various tools of structural biology — structural determination methodologies like NMR and X-ray crystallography and computational methodologies. It also offers protein production and biophysical characterization — all within one central resource. Investigators interested in how structural research might enhance their studies should contact Chazin, any of the other structural biology faculty, or one of the CSB assistant directors, Laura Mizoue, Ph.D., and Jarrod A. Smith, Ph.D. The current studies were supported by the National Institutes of Health.