Abstract 17379: Atrial Fibrillation Mutations in β3 Subunits Enhance DIII Voltage Sensing Domain Coupling to Channel Activation Which Inhibits Lidocaine Blockade
Background: Co-assembly of cardiac Na+ channels (Nav1.5) with β subunits modifies channel gating, expression, and post-translational modification. β subunit mutations have been linked to the Brugada and Long QT Syndromes, and atrial fibrillation (AF).
Hypothesis: We tested whether β3 subunits regulate Nav.1.5 ionic current and drug response by modulating the voltage sensing domains (VSDs).
Methods: The NaV1.5 α subunit contains four domains (DI-DIV), each with its own voltage sensing domain (VSD). We previously created four DNA constructs that carried a cysteine within a single VSD. Channels expressed in Xenopus oocytes and these cysteines were labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that tracked VSD conformation were simultaneously recorded using the cut-open configuration with and without β3.
Results: Steady state inactivation is significantly right shifted by β3(V1/2= -88.9 ± 1.1 SEM (with, +β3) and -97.8 ± 1.5 (without, -β3), p=0.002, n=4). β3 alsoright shifts DIII-VSD activation (V1/2= -93.0±2.3 +β3, V1/2= --114.8±0.8 -β3, p=0.001, n=4), while modestly left-shifting channel activation, suggesting enhanced DIII-VSD to pore coupling (V1/2GV-V1/2FV=55.0 ± 4.0 +β3, V1/2GV-V1/2FV=73.6 ± 2.4 -β3, n=4). DI and DII were not affected, while DIV was modestly shifted, consistent with DIII/DIV cooperativity. Extracellular domain AF-linked β3mutations, R6K and L10P, further enhance DIII-VSD to pore coupling (V1/2GV-V1/2FV=34.1 ± 5.8, (R6K), V1/2GV-V1/2FV=41.5 ± 4.7 (L10P), n=4). β3 nearly abolishes stabilization of the DIII-VSD by lidocaine (DIII FV shift by lidocaine: ΔV1/2lido=-27.71 ± 12.23 (+β3), ΔV1/2lido=-65.44 ± 3.83 (-β3), n=3). The conservative R6K mutation exacerbates this effect, suggesting a cation-pi interaction with NaV1.5. W1684 is co-localized with the DIII-VSD, and W1684A disrupted β3 modification of channel gating and the DIII lidocaine interaction.
Conclusions: β3modifies Nav1.5 gating by increasing DIII-VSD coupling to the pore via interaction with W1684. AF β3mutants further enhance DIII-VSD to pore coupling. The differential lidocaine response caused by WT and AF β3 mutants suggests a molecular mechanism whereby the lidocaine response is patient and heart-chamber specific.
Author Disclosures: W. Zhu: None. E.J. Hsu: None. B. Li: None. A.R. Schubert: None. Z. Varga: None. J.R. Silva: None.
- © 2015 by American Heart Association, Inc.