A comprehensive thermodynamic and structural characterization of ion channel function and its regulation by the lipid bilayer composition

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Project summary The central tenet of this grant application contains two interdependent components 1) how does the structure determine the function of K+-channels? and how does the cell membrane phospholipid composition regulate their structure-function correlations? Our work deal with these two fundamental questions, which encompasses three aspects of ion channel physiology: 1) Which are the structural changes underlying K+- channel gating, permeation, and selectivity? 2) How does a bidirectional allosteric coupling between the activation gate (AG) and the selectivity filter (SF) control K+-channels function? and 3) How does the cell membrane lipid composition regulate K+-channels behavior? Understanding at the atomic level how K+- channels work will assist during the discovery of novel therapeutic drugs. We will use several methodological advancements developed by us during the last 10 years of continuous funding from the NIH. These achievements are: 1) the elucidation of an atomic resolution gating cycle of a K+-channel 2) the engineering of a disulfide bridged locked open KcsA scaffold that produces atomic resolution diffracting crystals and allow us to characterize its function by electrophysiology at pH 7.0 (a physiologically relevant pH) 3) the measuring of the alkali metal ions binding affinity by Isothermal Titration Calorimetry of the whole selectivity filter of a K+-channel in the open conformation and/or of individual ion binding sites 4) the discovery of a novel mechanism of KcsA activation by reducing the thickness of the cell membrane and 5) the development of a new methodology for the overexpression of properly folded and functional Human K+-channels of Biomedical Importance in E. coli cells. Our work using KcsA as a structural surrogate is foundational of our current understanding of K+-channel function. Now we are expanding into human K+-channels by developing a groundbreaking new methodology for the overexpression of properly folded and functional human Kv-channels in E. coli cells, which eliminates the otherwise time consuming and outrageously expensive use of eukaryotic cells. We will develop an integrative understanding of how the structure of ion channels change its conformation to regulate their function within an energetic landscape determined by the lipid composition of the cell membrane. We aim to determine the structural changes underlying ion permeation, ion selectivity and C-type inactivation gating and their interdependence with the lipid bilayer composition of the cell membrane in a bacterial channel and in two human Kv-channels of Biomedical Relevance. Finally, we will produce a conceptual framework about how the allosteric coupling between a K+-channel’s selectivity filter and its activation gate define ion channel function and how is modulated by subunit cooperativity and the phospholipid composition of the cell membrane. The completion of this grant application will produce invaluable information to assist in the smart design of safer therapeutic drugs.