Abstract 13064: Voltage-Clamp Fluorometry Reveals a Unique Cardiac Phenotype for the hNaV1.5 DIII and DIV Voltage Sensors
Background: Inactivation of the human cardiac voltage-gated Na+ channel, hNaV1.5, is responsible for the refractoriness of the myocardium, modulating its ability develop and sustain arrhythmia. Functional channels are formed by monomers with four domains (DI-DIV) containing six segments (S1-S6) each. The positive charge-rich S4 acts as a voltage-sensor. Voltage-clamp fluorometry (VCF), which tracks conformational changes in environment around a cysteine-conjugated fluorophore, has not previously been used to study cardiac-specific S4 movements.
Methods: Fluorescence and currents were monitored simultaneously with cut-open VCF in Xenopus oocytes. Four novel constructs with cysteine substitutions in positions adjacent to S4 in each hNaV1.5 domain displayed voltage sensitive changes in fluorescence after being expressed and labeled with MTS-TAMRA.
Results: When depolarizing pulses were applied, DI, DII, and DIII activated rapidly (τ<1ms), while DIV moved slowly. Returning to negative potential, DII and DIII rapidly transited to the resting state (τ<1ms) without any observable delay. However, DIV and DI recovered slowly, suggesting that they may be immobilized by fast inactivation. The rate of DIV recovery increased with longer depolarizing pulses; once the pulse exceeded 56±9 ms, the recovery time constant decreased 37% (from 38±6 to 23±3 ms, n=7). Conversely, recovery from inactivation slowed (16%, n=3), when the pulse increased from 30 to 200 ms, implying a transient DIV S4 interaction with the inactivation particle.
Conclusions: In contrast with VCF results from the rat skeletal muscle isoform (rNaV1.4) that show an inactivation-linked DIII delay in recovery, our results show a rapid return and no delay, suggesting that DIII is not immobilized. DIV is significantly slower, as in rNaV1.4, however its immobilization appears to be only transiently linked to inactivation, indicating a short window when small molecules that interact with this sensor can target inactivation, beyond which inactivation likely involves either another segment within DIV or a different domain. Differences in S4 movements among rNaV1.4 and hNaV1.5 demonstrate variability in the link between inactivation and voltage sensing across NaV channel isoforms.
- © 2013 by American Heart Association, Inc.