Voltage-gated sodium (NaV) channels are fundamental signaling molecules in excitable cells that determine the rapid influx of Na+ ions. Perturbation to the function of human cardiac NaV1.5 channels (SCN5A gene) can result in arrhythmias and in some cases death. Genetic defects in the SCN5A gene are a known cause of long QT type 3 syndromes, and are associated with other life threatening conditions such as Brugada syndrome and sudden infant death syndrome. For normal functioning NaV channels, changes in intracellular calcium ([Ca2+]i) lead to fast inactivation of NaV1.5. This process is known to involve the intracellular calcium sensing protein calmodulin (CaM), specific elements of the channel located near the C terminus, and the linker region between domains 3 and 4 (D3D4 linker). Recent studies have confirmed the D3D4 linker functions as the fast inactivation gate (FIG). In several instances, the above-mentioned mutations occur in, or are likely to alter interactions and functioning of CaM and the FIG, which is the focus of this work. Recent advances in the characterization of CaM-FIG interactions have identified mutations to specific residues that uncouple fast inactivation gating from [Ca2+]i dependence. However, the molecular basis for the action of the Ca2+ sensing apparatus on the FIG is currently debated and largely unknown. Several models have been proposed in the literature based on conflicting views, but no functional model has been generated that is able to reconcile all the available data. I hypothesize that changes in [Ca2+]i lead to remodeling of CaM and specific elements in the C-terminus of NaV1.5, which result in CaM interacting with the FIG (D3D4 linker) in a manner that is NOT described by previous models. Understanding the molecular details of CaM-FIG interactions are essential to the development of an accurate model that describes the Ca2+ and CaM-dependent modulation of NaV1.5 fast inactivation. Completion of this project will provide a detailed description of the highly complex mechanism that involves CaM and the D3D4 linker translating changes in [Ca2+]i into modulation of fast inactivation gating of the human cardiac sodium channel. This investigation will utilize a complement of biophysical, structural, and electrophysiological techniques to characterize interactions between CaM and the FIG that are essential to this regulatory mechanism. The results will be used to determine the relative extent of perturbation to NaV1.5 fast inactivation caused by mutations to the CaM-FIG complex. This information will aid in understanding the molecular basis of specific cardiac life threatening arrhythmias as well as lay a broad foundation for evaluating the potential use of small molecules to target the NaV1.5 [Ca2+]i sensing apparatus for the treatment of certain cardiac arrhythmia syndromes.

Public Health Relevance

Perturbation to the function of human cardiac sodium ion channels can result in arhythmias and in some cases death. Completion of this project will aid in understanding the molecular basis of specific cardiac life threatening arrhythmias as well as lay a broad foundation for evaluating the potential use of small molecules to target the NaV1.5 [Ca2+]i sensing apparatus for the treatment of certain cardiac arrhythmia syndromes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32HL117612-01
Application #
8456784
Study Section
Special Emphasis Panel (ZRG1-F04-D (20))
Program Officer
Wang, Wayne C
Project Start
2014-02-01
Project End
Budget Start
2013-08-01
Budget End
Support Year
1
Fiscal Year
2013
Total Cost
$49,214
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Makita, Naomasa; Yagihara, Nobue; Crotti, Lia et al. (2014) Novel calmodulin mutations associated with congenital arrhythmia susceptibility. Circ Cardiovasc Genet 7:466-74
Hwang, Hyun Seok; Nitu, Florentin R; Yang, Yi et al. (2014) Divergent regulation of ryanodine receptor 2 calcium release channels by arrhythmogenic human calmodulin missense mutants. Circ Res 114:1114-24