Perturbation of sodium channel structure by an inherited Long QT Syndrome mutation

Abstract

The cardiac voltage-gated sodium channel (NaV1.5) underlies impulse conduction in the heart and its depolarization-induced inactivation is essential in control of the duration of the QT interval of the electrocardiogram (ECG). Perturbation of Nav1.5 inactivation by drugs or inherited mutation can underlie and trigger cardiac arrhythmias. The carboxy terminus plays an important role in channel inactivation, but complete structural information on its predicted structural domain is unknown. Here we measure interactions between the functionally critical distal C-T alpha helix (H6) and the proximal structured EF hand motif using transition metal ion FRET. We measure distances at three loci along H6 relative to an intrinsic tryptophan, demonstrating the proximal-distal interaction in a contiguous carboxy terminus polypeptide. Using these data together with the existing NaV1.5 carboxy terminus NMR structure, we construct a model of the predicted structured region of the carboxy terminus. An arrhythmia associated H6 mutant which impairs inactivation decreases FRET, indicating destabilization of the distal-proximal intramolecular interaction. These data provide a structural correlate to the pathological phenotype of the mutant channel.

Accurate High-Throughput Structure Mapping and Prediction with Transition Metal Ion FRET

“…From our data, we conclude that using smaller dyes such as bimane results in more accurate measurements of backbone distances in proteins without the need for complex modeling of fluorophore positions. Similar results should be obtained from native small fluorophores such as tryptophans (Glaaser et al, 2012). Both bimane and tryptophan, however, are fairly dim fluorophores.…”

“…In tmFRET, a colored transition metal ion (Ni 2+ or Cu 2+ ) acts as an energy acceptor for small organic fluorophores such as bimane and tryptophan (Glaaser et al, 2012; Taraska et al, 2009a; Taraska et al, 2009b). Because metals have extremely low extinction coefficients, they have very short R 0 values and thus act as energy acceptors only over very short distances (Taraska et al, 2009a; Taraska et al, 2009b).…”

Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

“…Electrophysiological studies of I Na in Na V 1.5 COOH-terminal (including the IQ motif) LQTS mutants reveal I Na,L potentially related to perturbed interactions of the COOHterminus with the III-IV loop, i.e., fast inactivation gate (37,54). This might explain the arrhythmogenic phenotype in inherited mutations involving the IQ motif (and possibly also the III-IV loop).…”

A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels

“…tmFRET is a FRET technique that uses small, covalently attached fluorophores as donor molecules and colored transition metal ions (e.g. Ni 2ϩ and Co 2ϩ ) bound to pairs of histidine residues as acceptors (6,(23)(24)(25). This method can report very short distances (8 -20 Å), virtually eliminates the orientation dependence of classical FRET, and allows the experimenter to tune the dynamic range of distance measurements by changing the acceptor ion.…”