Fluorescence Fluctuation Spectroscopy to Detect Interactions Between Dopamine Receptors and Calcium Channel in Pancreatic β-Cells

Abstract

Dysregulation of glucose homeostasis can lead to the development of diabetes, and secretion of insulin from pancreatic β-cells plays a key role in maintaining proper blood glucose levels. Previous research has shown that the neurotransmitter dopamine can inhibit secretion of insulin from β-cells by activating the dopamine receptor D3 (DRD3), which reduces the amplitude and frequency of intracellular free calcium oscillations (Ustione & Piston, Mol. Endocrinol.26, 1928 (2012)). To target this dopaminergic feedback loop for treatment of diabetes, the signaling pathway downstream from the dopamine receptor to calcium flux must be better understood. We hypothesize that after dopamine activation of DRD3, the Gβγ complex is released and interacts directly with voltage-gated calcium channels. To test this hypothesis, we are utilizing fluorescence fluctuation spectroscopy to determine the dynamic localization distributions and interactions between different proteins. Two-color fluctuation analysis allows us to correlate the diffusion and kinetics of two proteins of interest without the constraints of FRET. Thus, expressing the proteins of interest tagged with a fluorescent protein in stable β-cell lines provides an in vitro method to study the signaling pathway. Details of the kinetic localization and interaction rates of the G-protein coupled DRD3, the Gβγ complex, and the Cav1.2 calcium channel subunit will be presented. In addition, we will show the changes in protein localization and correlation due to dopamine stimulation. Molecular interactions of proteins due to dopamine stimulation illustrate the method of which the dopaminergic feedback loop works. This provides the necessary information for using the dopaminergic feedback pathway as a method to inhibit insulin secretion, and thus as a potential therapeutic target for the treatment of non-insulin dependent diabetes.