Protein semi-synthesis enables real-time optical tracking of intracellular conformational changes during sodium channel inactivation
Dynamic conformational changes in intracellular domains fundamentally affect the function and pharmacology of many membrane proteins. For example, sodium influx through the cardiac voltage-gated sodium channel (NaV1.5) is rapidly terminated through conformational changes that result in pore closure, a transition known as inactivation. Inactivation involves NaV1.5 intracellular regions,…
Sodium channel NaV1.5 undergoes rapid conformational changes upon influx of sodium, which are essential for its function and pharmacology. Inactivation, a crucial process terminating sodium influx, involves specific intracellular regions, particularly the DIII-DIV linker. Dysfunction of this region contributes to cardiac arrhythmias.
However, studying the conformational changes and their modulation by auxiliaries or drugs in live cells has been challenging due to limitations in labeling techniques. In this study, researchers employed a novel method of combining live-cell protein semi-synthesis with voltage-clamp fluorometry (VCF) to track the intracellular dynamics of NaV1.5 in real-time.
This approach allowed them to monitor both fast and steady-state inactivation processes and their voltage dependence. Additionally, the study revealed that both lidocaine and auxiliary proteins influenced the kinetics of conformational changes in the DIII-DIV linker. By demonstrating the effectiveness of this combined technique, the researchers have provided a powerful tool for dissecting complex intracellular conformational changes in membrane proteins.
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