PO-01-206 MOLECULAR MODELING OF CLINICAL KCNJ2 MUTATIONS REVEALS STRUCTURE-FUNCTION IMPACT ON PIP2 BINDING SITE

Binding phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for Kir2.1(encoded by KCNJ2), function. Disease associated KCNJ2 mutations, such as those with Andersen-Tawil Syndrome (ATS), may exert molecular dysfunction interrupting PIP2 binding, however a full-length model of Kir2.1 has not been developed to explore this hypothesis. Elucidate the structure and determine functional impact clinical mutations on binding using experimental improved dynamic (MD) method. Whole-cell patch clamp experiments WT-Kir2.1 selected were performed stably transfected HEK293 cells standard protocol. A tetramer (open closed conformational states) based Kir2.2 cryo-EM MOE was created. Mutations (R67Q, G300D, R218L) individually introduced channel, mutagenesis analysis performed. Initially, MD simulation 100ns wild-type (WT) mutated structures (GROMACS software). We expanded include +/- bound PIP2. Due > 77% degree homology (6m84) Kir2.1, we protocol test system. The 2.5% free molecules released into membrane 700ns Amber software. Homomeric WT demonstrated normal function while R67Q, R218L showed complete loss Mutational revealed that residue changes result in disruption hydrogen bonding affects structural conformation at sight caused fluctuation residues other regions, i.e., selectivity filter pore domain. validated sites. Moreover, found mutation bonds hydrophobic interaction between channel significant changes. first full length ATS disrupt bonding. Further, sites which illustrate robustness our method, technique can be applied delineate Combined validation, modeling will increase understanding structure-function relationships mutations.