UNCOVERING THE ROLE OF SPCA2 IN THE SK3 SIGNALING COMPLEX

Ca2+ ions govern a wide range of essential cellular functions. A highly complex cooperative network of Ca2+ signaling components maintains healthy cell functions. A defect in a single Ca 2+ signaling protein can lead to an abnormal cellular Ca 2+ milieu and cause devastating long-term consequences such as carcinogenesis. We are particularly interested in a Ca 2+ entry pathway that activates K+ efflux through Ca2+-activated K+ (KCa2+) channels. This leads to hyperpolarization of the membrane, enhancing the driving force for Ca2+ influx as part of a positive feedback mechanism. Importantly, such an interplay of Ca2+-permeable and Ca2+-activated ion channels can trigger or promote the development of cancer cells. In this project, we focus on the co-regulation of the small conductance K+ channel, SK3, a Ca2+ ion channel, Orai1, and a Ca2+-ATPase in the Golgi secretory pathway, SPCA2. Breast and colon cancer cell migration is promoted by the co-regulation of SK3 and Orai1 by elevating cytosolic Ca 2+ levels and increasing K+ efflux via SK3 channels. SPCA2 induces Ca2+ influx via Orai1 in breast cancer cells, thereby triggering tumorigenesis. This project aims to study the role of SPCA2 in regulating the interplay of SK3 and Orai1 channels. Although there is clear evidence for a co-regulation of SK3 and Orai1, the molecular linkers for this interplay are still poorly understood. We propose that SPCA2 represents another suitable candidate to regulate the SK3 signaling complex. We will study for the first time the functional impact of SPCA2 on the SK3 channel alone as well as on the SK3-Orai1 complex and uncover important molecular determinants thereof using a combined approach of functional, fluorescence microscopy, and biochemical studies. In addition to the current knowledge based on the findings in breast cancer cells, we will expand our investigation to study the molecular interaction of this trio complex in melanoma and prostate cancer-derived cells. We expect to identify a direct molecular link that stabilizes the SK3-Orai1 complex. Identification of these targets (SK3, SPCA2, Orai1) and regulatory mechanisms and investigating their molecular interaction helps, in the long run, to establish strategies for therapeutic developments that directly interfere with the communication of SK3, SPCA2, and Orai1. Hence, our approach will not only allow for an in-depth understanding of a disease-relevant branch in Ca2+ signaling but can, in consequence, provide a fundamental basis for the examination of the trio-complex in primary cancer cells and cancer biopsies.