Dissertation/Thesis Abstract

Structural basis of cardiac voltage-gated sodium channel inactivation
by Glaaser, Ian W., Ph.D., Columbia University, 2010, 118; 3447868
Abstract (Summary)

Channelopathies, a set of diseases caused by mutations in genes coding for ion channels or ion-channel associated proteins, are an increasingly important class of human disorders. Disease-linked mutations have been identified in several voltage-gated sodium channel isoforms, and functional characterization of these mutants has shown that a significant portion of them results in impaired inactivation. A cluster of these mutations associated with the congenital Long QT syndrome (LQTS) has been identified and characterized in the carboxy terminus of the cardiac voltage-gated sodium channel (Nav1.5), implicating a region previously not associated with playing a role in channel gating. A structural model of the Nav1.5 carboxyl terminus (C-T) based on homology to the amino-terminal lobe of Calmodulin predicts six alpha helices (H1-H6), the first four forming two EF hand pairs and the last helix (H6) containing the CaM binding IQ motif. Studies presented here using whole cell patch clamp and tryptophan fluorescence suggest a role of the putative interface between H1 and H4 in control of channel inactivation and stability of the carboxy terminal structure. Tryptophan fluorescence experiments to test for the environment of the sole tryptophan residue in the carboxy terminus are consistent with model predictions. The results also indicate that mutation of hydrophobic residues integral to the H1-H4 interface disrupts protein stability and markedly alters channel inactivation, providing evidence that stabilization of the C-T structure via the H1-H4 hydrophobic interface is necessary to preserve physiologically essential inactivation of the Nav1.5 channel. Subsequently, NMR structural models of the proximal C-T from two voltage-gated sodium channel isoforms confirmed the computational model predictions and the presence of the hydrophobic interface between H1 and H4.

Neither the NMR structures nor the computational models were able to predict the location of the most distal helix (H6). The sixth helix plays a critical role in normal inactivation of the alpha-subunit, in addition to providing a binding motif for calmodulin. Using a modification of transition metal ion FRET, interactions were observed between the EF hand motif (H1-H4) and the IQ motif (H6) in cis. Experiments designed to test for the consequences of a disease-linked mutation on this interaction found that introduction of a LQT3 mutation on H6 disrupted this interaction. These studies provide insight into mechanisms through which structural motifs in voltage-gated sodium channels C-terminus modulate channel inactivation.

Indexing (document details)
Advisor: Kass, Robert S.
Commitee:
School: Columbia University
School Location: United States -- New York
Source: DAI-B 72/05, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Physiology
Keywords: Channel inactivation, Ion channels, Long QT syndrome, Sodium channels, Voltage-gated sodium channels
Publication Number: 3447868
ISBN: 9781124533155
Copyright © 2019 ProQuest LLC. All rights reserved. Terms and Conditions Privacy Policy Cookie Policy
ProQuest