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The basis for calcium regulation of the cardiac sodium channel

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Title: The basis for calcium regulation of the cardiac sodium channel
Author: Sarhan, Maen Fuad
Degree Doctor of Philosophy - PhD
Program Pharmacology and Therapeutics
Copyright Date: 2012
Publicly Available in cIRcle 2012-09-28
Abstract: Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are potently modulated by cytoplasmic calcium ions resulting in an increase in the availability of channels available for a depolarization. Characterization of calmodulin (CaM) and Ca²⁺ binding to the C-terminus have failed to identify a mechanism due to data which is inconsistent and difficult to interpret. We examined the possibility of whether CaM interactions with the inactivation gate of the sodium channel between domains three and four (DIII-IV linker) and C-terminus could possibly explain how the channel is modulated by Ca²⁺. We employ highly purified recombinant proteins for X-ray crystallography and Isothermal titration calorimetry (ITC) to identify tyrosine 1494 as an aromatic anchor for the C-terminal lobe of Ca²⁺/CaM binding to the DIII-IV linker. Through crystallographic insight, and confirmation by ITC, we incorporate mutations in the DIII-IV linker that enhance or diminish Ca²⁺/CaM binding, which sensitize or abolish Ca²⁺ regulation of full-length channels in whole-cell electrophysiological experiments. The single lobe interaction with the DIII-IV linker opens the possibility that the N-terminal lobe interacts with another region of the sodium channel. To examine this possibility we used ITC to examine how CaM interacts with the C-terminus and found that in the absence of Ca²⁺, CaM interacts with its C-lobe with very high affinity, allowing it to act as a resident Ca2+ sensor. As Ca²⁺ levels rise, lobe switching occurs, and preferential N-lobe binding to the C-terminus followed by C-lobe binding to the DIII-IV linker. The Ca²⁺/CaM DIII-IV crystal structure we obtained harbors the positive of five disease mutations involved in deadly arrhythmias. We find that two of these mutations altered both Ca²⁺/CaM binding as well as Ca²⁺ regulation in full length channels suggesting that calcium dysregulation may be involved in cardiac arrhythmia. We conclude that Ca²⁺ regulation of the cardiac voltage gated sodium channel occurs through CaM bridging of the C-terminus and the inactivation gate and that dysfunction of this process may result in cardiac arrhythmia.
URI: http://hdl.handle.net/2429/43308
Scholarly Level: Graduate

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