Project Summary Big potassium (BK) channels, named after their "Big K+" conductance, are atypical potassium channels activated synergistically by voltage and calcium. Expressed in nearly all organs, BK channels have important roles in the function of the nervous, gastrointestinal, reproductive, visual, endocrine, urinary, lymphatic, skeletal muscle, and cardiovascular systems. BK channel activation requires the allosteric binding of calcium to the cytoplasmic C- terminal of the channel. Therefore, spatial localization at the plasma membrane and proximity to calcium sources are two critical factors regulating BK channel function. My previous work using super-resolution microscopy has demonstrated that rather to be homogeneously distributed at the plasma membrane, BK channels organize into large clusters. In addition, I demonstrated that these BK channel clusters organize into a higher hierarchical arrangement with voltage-gated calcium channels (CaV), which are their primary calcium source for activation. Combining electrophysiology and pharmacology, I established that this BK-CaV co-clustering is key for the activation of BK channels at physiological hyperpolarized voltages. Yet, the molecular mechanisms that mediate BK channel cluster formation and the co-clustering with specific CaV channels remain mysterious. Research in my lab now builds on my discoveries and focus on identifying the molecular mechanisms behind BK-BK and BK- CaV channel clustering and the role of clustering in the fine-tuning of BK function. Leveraging our expertise in super-resolution microscopy, quantitative fluorometry, ion channel biophysics, and lipid metabolism, we have identified three research areas that will advance our understanding of the mechanisms behind BK channel clustering. Research area 1 will focus on the study of the mechanisms mediating the formation and maintenance of BK-BK clusters – for this, we will systematically evaluate the role of the BK channel C-terminal and the role of BK β and γ auxiliary subunits. Research Area 2 will focus on the mechanisms mediating BK-CaV channel co- clustering – for this, we have identified AKAP150 and RIB2 as two strong candidates that interact with both channels and can underlie the co-clustering. Research Area 3 will focus in a poorly explored topic, the role of membrane lipid composition in the clustering of BK-CaV channels – I will leverage my expertise in the study of ion-channel modulation by phosphoinositides and cholesterol to lead the research on how these two signaling lipids are involved in ion channel clustering. Overall, this research program is designed to provide new insights into how BK channel clusters are formed and maintained near calcium sources. In the long-term, this proposal will open new avenues for the study of mechanisms modulating BK channel function in health and disease.