Mapping the Structure of the Cardiac Voltage-Gated Sodium Channel (Nav1.5) C-Terminus: Implications of An Lqt3 Mutant

Abstract

The cardiac voltage-gated sodium channel(Nav1.5) underlies the upstroke of the cardiac action potential and can contribute to action potential duration. Defects in sodium channel inactivation, for which the carboxy terminus(C-T) plays an important role, are linked to cardiac arrhythmia. Studies of the C-T identify four proximal helices that form an EF hand motif and two predicted distal helices whose location relative to the EF hand motif are unknown. We used transition metal ion FRET to measure intramolecular interactions within the C-T to locate the predicted distal helix(H6). A single intrinsic tryptophan in the 1st C-T helix(H1), and Ni2+ coordinated by engineered di-histidine residues were used as the fluorescence donor and acceptor, respectively. Initial experiments confirmed the validity of the use of Trp as the donor and measured the distance between the Trp and a di-histidine on the 4th helix consistent with the NaV1.5 EF NMR (16.7±0.3 vs. 16.8A). Next, a distance between the Trp and a di-histidine on the middle of H6 was measured, demonstrating this interaction for the first time in cis. Introduction of an LQT3 mutation, S1904L, previously shown to disrupt inactivation, in the H6 di-Histidine background increased the distance such that there was no FRET. This indicated that the di-Histidine had moved >5 A. The change in distance correlates with disruption of inactivation, suggesting that the H6 interaction with the EF hand domain may be necessary for proper inactivation. Finally, we were able to measure distances for additional sites on H6 providing information about the orientation and distance of H6 relative to the EF hands. These data provide a structural framework within which mutational effects can be examined and provide a structural correlate to altered function in an LQT3 mutant channel.