PROJECT SUMMARY Cell to cell communication is critical for function in all multicellular organisms. A key factor for intercellular communication is regulation by Ca2+ concentration. Ca2+/calmodulin dependent protein kinase II (CaMKII) is a Ca2+ sensitive enzyme that is encoded by four genes in mammals: α, β, γ, and δ. There is an incredible amount of diversity generated from the four vertebrate CaMKII genes. Alternative splicing produces up to 386 transcripts, which leads to the production of 386 proteins that are then differentially post-translationally modified, and mix to form hetero-oligomeric complexes, ultimately culminating in thousands of chemically distinct CaMKII proteoforms. We are specifically interested in the crucial roles CaMKII plays in long-term memory formation (neurons: α, β), fertilization (oocytes: γ), and cardiac physiology (cardiomyocytes: δ). Intriguingly, these cells all communicate using Ca2+ oscillations but on vastly different timescales (minutes to milliseconds). How does one enzyme accommodate this multifunctionality? We hypothesize that selective splicing and modification creates a unique set of CaMKII variants expressed in specific cell types, thereby leading to differential functional outputs. Fully elucidating these complex biological roles requires a deeper understanding of CaMKII variation at the sequence and protein level, structural and conformational ramifications of these variations, and how these variables affect CaMKII interactions within the cell. In this proposal, we seek to expand our understanding of CaMKII function inside cells using a combinatorial approach of sequencing, biochemistry, structural biology, and cellular assays. Completion of the proposed work will allow us to uncover the molecular basis for the many roles of CaMKII in neurons, cardiomyocytes, and oocytes – with far-reaching implications on therapeutic intervention for neurologic disease, cardiac dysfunction, and infertility.