Engineering Next-Generation Tools to Dissect Calcium Channel Signaling in Pancreatic Beta Cells

Ca2+ influx through voltage-gated calcium channels (VGCCs) is necessary for glucose-stimulated insulin secretion in pancreatic β-cells (PβCs). VGCCs are thus a potential locus for both PβC-dependent pathophysiology and therapy. CaVβ subunits (β1-β4) are powerful regulators of VGCCs by controlling α1 subunit trafficking and tuning channel gating. A central unresolved question is: how does Ca2+ influx via VGCCs give rise to divergent functions in PβCs such as insulin secretion and excitation-transcription coupling? The PI hypothesized that in PβCs distinct CaVβs are instrumental in organizing VGCCs into discrete macromolecular complexes with specialized functions. A significant barrier to rigorously assess the functional roles of CaVβ molecular diversity in excitable cells, including PβCs, is the inability to inhibit VGCCs based on the identity of their resident CaVβ. The PI will develop novel genetically-encoded CaV channel blockers that enable inhibition of CaVβ-specific VGCC complexes, and apply them to decipher signaling functions of CaVβs in PβCs. The approach exploits a bioengineering method to generate genetically-encoded VGCC inhibitors by pairing the specificity of single-domain antibodies (or nanobodies) with the consequential catalytic activity of an E3 ubiquitin ligase. Using Cavβ isoform-selective nanobodies with molecular biology, electrophysiology, flow cytometry, fluorescence resonance energy transfer (FRET), ion channel engineering, and biochemistry, the PI will develop and engineer nanobodies to selectively inhibit VGCCs on the basis of their resident CaVβ, and elucidate the functions of CaVβ specific VGCC complexes in pancreatic β-cells.