RII Track-4:NSF: Unravelling Gating Mechanisms of Ion Channels Using Computational and Experimental Ultrafast Vibrational Spectroscopy

K+ channels are membrane proteins that facilitate the transport of K+ ions across the cell membranes. They play key roles in nerve and muscle relaxation, cognition, and regulation of blood pressure. While K+ channels have been extensively studied over many years, the mechanism of activation of ion channels remains a topic of ongoing debate that has not been possible to solve with established experimental methods.

Two-dimensional infrared (2D IR) spectroscopy is an emerging analytical technique that probes structural changes of proteins with chemical bond specific spatial and high temporal resolution. Recent technological advances permit applications of 2D IR spectroscopy to ion channels under physiologically realistic conditions. This project combines molecular dynamics simulations with computational spectroscopy to study activation mechanisms of K+ channels.

Simulations will be validated by 2D IR experiments and, in turn, will be used to design new experiments that can provide most clear insight into activation mechanisms of K+ channels. The proposed work will derive the atomistic-level description of function of K+ channels. Understanding the mechanisms underlying K+ channels functioning is a key factor in determining the cause of severe diseases such as cardiac arrhythmias and epilepsies. It offers the prospect of designing therapies for ion channel pharmacology.

This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4) project would provide a fellowship to an Assistant Professor at the University of Delaware (UD). Voltage-gated K+ channels (Kv) are integral membrane proteins that selectively conduct K+ ions across cell membranes according to the electrochemical gradient. The voltage-sensing domain (VSD) of Kv senses changes in the membrane electrical potential and triggers a conformational change resulting in the opening and closing of the channel.

Despite a wealth of NMR and X-ray crystallography data, many fundamental questions regarding the function of the VSD remain open. It has been hypothesized that the S4 helical segment of the VSD undergoes a conformational and/or hydrational change during voltage-gating, but this hypothesis has never been tested by a direct structural measurement because most biophysical structural techniques cannot be performed under applied voltage. 2D IR spectroscopy can probe protein structures with site-specific resolution and under physiological conditions including applied voltage.

It can also probe the gating dynamics of the VSD occurring on the millisecond timescale. Recently the sensitivity of 2D IR spectroscopy has been significantly increased, making it uniquely suitable to study ion channels. The proposed work will combine molecular dynamics simulations with computational 2D IR spectroscopy to elucidate the conformational and hydrational changes of the VSD during voltage activation.

The existing structural models of the VSD will be simulated and tested against 2D IR experiments. Based on the comparison with experiments, the models will be refined, or new models will be developed. Simulations will also be utilized to design new 2D IR experiments.

We aim to determine the relevant conformations of the VSD and the order they occur during voltage activation.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.