K+ channel structural dynamics landscape: from selectivity to gating

About This Grant

K+ channel structural dynamics landscape: from selectivity to gating The chemical and electrical activities that make life possible are controlled by a constellation of ion channels. These are transmembrane proteins that regulate the differential concentration of ions across the cell membrane, with, in the case of K+ channels, the extraordinary ability to differentiate between extremely similar ions such as Na+ and K+. Although ion selectivity and gating mechanisms have been studied for many years, the underlying structural temporal dynamics remain poorly understood. Using techniques such as single molecule FRET (smFRET) or fluorescent non-canonical amino acids incorporation, it is possible to assess ion channel intramolecular movements and states in real time. In this project, I intend to incorporate a wide range of techniques, from electrophysiology to single molecule approaches to delineate the real-time dynamics of K+ channels in synthetic and live cell membranes. By combining my prior experience in ion channel biophysics and electrophysiology, along with establishing new approaches and training in relevant techniques, including smFRET, hyperspectral imaging, structure determination and Molecular Dynamic (MD) simulations, I propose two specific aims to address the unifying question: What are the structural dynamics of ion selectivity and gating in K+ channels? These studies will, first, test the hypothesis that the potassium channel selectivity filter (SF) with sequence TxGYG becomes dynamic and unstable in the absence of K+, acquiring more stable conformations in the presence of K+, using NaK/NaK2K (non-selective and K+ selective) channels as models. This aim, giving new insights about ion channel selectivity, will be completed during the K99 phase. Development of additional techniques and theoretical skills will allow me to broaden my efforts in the R00 phase to the study of structural dynamics of channel gating mechanisms in more complex channels, including the pharmacologically relevant eukaryotic Two-Pore-Domain K+ channel family (K2P) that contributes to leak currents in excitable and nonexcitable tissues. This K+ channel family plays a critical role in a multitude of physiological and pathological conditions and understanding the gating and selectivity mechanisms will help to modulate their behavior to overcome different pathologies. Overall, the proposed integrated research and training will facilitate my becoming an independent researcher using a wide range of cutting-edge techniques to assist in the better understanding of membrane protein structural dynamics in health and disease. Project Summary/Abstract