Abstract 13319: Distinct Mechanism of Lidocaine Interaction With the DIII Voltage Sensor of the Cardiac Sodium Channel
Background: The human voltage-gated Na+ channel, hNaV1.5, which initiates the cardiac action potential, can be formed by monomeric α subunits containing 4 domains (DI-DIV), each with 6 membrane-spanning segments (S1-S6). Class Ib anti-arrhythmics, such as lidocaine, are widely used for therapy and target hNaV1.5. Here, we test whether a recent technique, voltage clamp flourometry (VCF), can be used to assess details of the lidocaine interaction with the DIII voltage sensing mechanism of hNaV1.5.
Methods: NaV voltage sensing is accomplished by positively charged residues residing in S4 of each domain. For VCF recordings, a cysteine was introduced into the DIII S3-S4 linker. mRNA from this construct was injected into Xenopus oocytes, and labeled with TAMRA-MTS fluorophores after 3-5 days. Fluorescence from TAMRA-MTS, conjugated to the engineered cysteine in the DIII S3-S4, could then be observed to directly track the DIII-S4 position. Fluorescence signals and currents in the with and without 5 mM lidocaine were recorded using the cut-open VCF technique at 19 °C to resolve fast kinetics.
Results: A reproducible 50% reduction in the change in fluorescence magnitude following a depolarizing pulse was observed, which correlated with current block. This reduction implies complete immobilization of the DIII-S4 segments by lidocaine, preventing the DIII sensor from returning to its resting state. Comparison of normalized fluorescence magnitude vs voltage (F-V) before and after lidocaine application showed no significant difference, suggesting that the DIII-S4 moves normally in un-blocked channels and is fully immobilized by lidocaine.
Conclusion: Previous results in the skeletal muscle isoform showed a dramatic shift toward negative potentials in the DIII F-V with lidocaine. We show appropriate channel block with lidocaine in the cardiac isoform without the leftward DIII-S4 shift but with significant immobilization, implying a unique interaction with the cardiac Na+ channel. The ability to discern distinct consequences of lidocaine block between the skeletal muscle and cardiac Na+ channel isoforms demonstrates the potential of VCF methodology for characterizing molecular mechanisms of anti-arrhythmics that target hNaV1.5.
- © 2013 by American Heart Association, Inc.