Project Summary M channels are critical for regulating the excitability of neurons. Dysfunction of M channel activity can cause epilepsy. While M channels have been intensely studied, the interplay of several G-protein-regulated signaling cofactors on these channels is still poorly understood. M channels are hetero-tetrameric pore structures formed by the combination of subunits Kv7.2-5. Over 80 mutations have been mapped to the Kv7.2 subunit, being a primary cause of neonatal epilepsy. Many of these mutations lie within the binding domains for at least three critical signaling cofactors: calmodulin (CaM), phosphatidylinositol 4,5-bisphosphate (PIP2) and protein kinase (PKC). How PIP2 and CaM binding to Kv7.2 harmonize to fine tune channel activity is obscure. Moreover, the role played by PKC in tuning this binding is obscure. My preliminary data shows that PIP2 and CaM may simultaneously bind the B helix with phosphorylation tempering this binding. My NMR studies so far show that phosphorylation reconfigures the apoCaM-B helix interaction, suggesting the interplay between these cofactors has significant impact on channel structure. I will test the overarching hypothesis that phosphorylation at S520 and S527 fine-tunes the ability of PIP2 and CaM to bind to Kv7.2 and control M channel activity. This hypothesis will be tested using three aims and will provide mechanistic understanding of how phosphorylation, and dependent PIP2 and CaM binding affects the structure of Kv7.2 to control channel activity. In Aim 1, I will use advanced 3D and 4D NMR to resolve the solution structure of purified Kv7.2 C terminus. Aim 2 will use these NMR spectra to define the affinity of PIP2 to Kv7.2 and describe the stoichiometry and mode of binding between PIP2 and the multiple sites on Kv7.2. Aim 3 will elaborate how phosphorylation within the B helix directs the interplay between CaM and PIP2 binding to Kv7.2. The proposed study is innovative because it will address a longstanding question about how and where PIP2 binds Kv7.2, and will elaborate how phosphorylation directs the interplay between CaM and PIP2 as they bind Kv7.2. My training in advanced 3D and 4D NMR will be critical for my career advancement as I plan to use this rigorous technique throughout my career. The rich resources and supportive environment at UT Health combined with my expertise in biophysical methods on ion channels makes me the ideal candidate to study the mechanisms underlying the regulation of M channel activity. The MOSAIC career award will provide valuable financial support to help me begin my multidisciplinary research program focused on elucidating the molecular mechanisms of ion channel regulation.