Project (3): K⁺ Signaling dynamics of Na⁺/Ca²⁺-activated K⁺ channel complexes in health and disease

PI: Robert Lukowski

Co-worker: Lucas Matt, Ying Zhang

It has been widely recognized that the Ca²⁺-activated K⁺ channel (KCa) of big (BK, Slo1, MaxiK) and intermediate (IK, KCa3.1) are involved in neuronal-, cardio-vascular, metabolic and immune functions and dysfunctions. In human diseases, changes in expression levels, altered configurations of the KCa ion channel complexes and their activity have been identified, indicating that they represent promising targets for future drug therapies of hypertension, obesity, autoimmune diseases and cancer. Our available gene-targeted KCa mouse models are insufficient to fully explore the cellular mechanisms by which IK, BK and their respective partner proteins affect the pathophysiology of these disorders. Since the role of ion channels critically depend on their integration into protein complexes, a comprehensive approach towards the identification of cell specific KCa signalling complexes in native tissues at the patho-/physiological level would be desired. Hence, novel strategies and tools are needed for future studies. Herein, we aim to establish a highly versatile system, which is based on a combination of state-of-the-art BAC transgenesis, small protein anchors and our previously introduced KCa-deficient mouse lines. The BAC-driven expression of un-/modified channels will permit a highly cell type specific reconstitution of the KCa proteins on a BK- or IK-negative mouse background. Using this strategy it is one aim of this project to rescue given disorders of the mutant mouse lines, which closely resemble specific aspects of the related human diseases. At the same time, the attached Strep/FLAG tandem affinity purification (SF-TAP) epitope will allow an efficient enrichment and the rapid isolation of high-purity channel complexes for the proteomic analyses by LC-MS/MS. Together, these studies should uncover the cell-context specific signalling networks defined by the BK and IK, the dynamic changes of their interactomes, and post-translation modifications of the channels providing important clues about their role in biology and pathophysiology.

In a complementary approach we use innovative Förster resonance energy transfer (FRET)-based K⁺ -binding probes, which allow us to perform reliable and reproducible measurements of K⁺ dynamics within living cells, various extracellular compartments and living animals. These K⁺-biosensors called GEPIIs will help us to understand if and how pharmacological modulators of K⁺ channels affect global and sub-/cellular K⁺ dynamics and how these dynamics correlate with different cell functions in health and disease (Bischof et al., 2017).