2D IR spectroscopy of voltage gating in potassium ion channels

University Of Wisconsin-Madison, Madison WI

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Abstract

Abstract Voltage-gated potassium ion channels (Kv) are responsible for reestablishing the resting potential of cells upon membrane depolarization. They consist of two domains, a voltage sensing domain (VSD) and a pore domain. The voltage-dependence of the VSD is sensed by the positively charged TM4 helix, causing a translation and/or secondary structure change that tugs on the pore domain to open the channel. There exist many high-resolution atomic structures of Kv channels in the depolarized state, but since it is experimentally difficult to obtain structural information under an electric field, most of the information about the polarized state comes from mutated proteins that approximate the hyperpolarized structure and from molecular dynamics simulations. During the last round of funding, we developed a method for measuring two-dimensional infrared (2D IR) spectra of membrane- embedded proteins under an electric field. We did so by using surface enhanced plasmons to increase the sensitivity of 2D IR spectroscopy by many orders of magnitude, making possible measurements on a single bilayer of membrane-embedded proteins across which a potential can be applied. Working with the Valiyaveetil lab, we have now measured voltage-dependent structural changes in the VSD of KvAP using cyano-labeled amino acids incorporated into the TM4 helix with in vivo nonsense suppression. In collaboration with the Kananenka group, we simulated the local electric field on the cyano group from molecular dynamics simulations, thereby relating the structural features of the hyperpolarized VSD to the experimental measurements. We also developed a sample cell that can create a fast voltage jump, enabling successive 2D IR spectra to measure the structural kinetics of the proteins as they switch from the depolarized to hyperpolarized state (or vice versa). In Aims 1 and 2 of this proposal, we will apply this methodology to study the voltage-dependent structural changes of the VSD from KvAP and Shaker, respectively, as well as in the full ion channels. In Aim 3, we will further develop and apply voltage-triggered 2D IR spectroscopy to time-resolve their structural motions. This combination of 2D IR spectroscopy, cyano tags or isotope labeling, and molecular dynamics simulations is a new means of investigating the structural dynamics of the hyperpolarized states of potassium ion channels and other membrane proteins.