Determining the Dopaminergic Feedback Pathway in Pancreatic β-Cells with Fluorescence Fluctuation Spectroscopy

Abstract

Tight regulation of insulin and glucagon, two hormones secreted by the pancreatic islet, allows the body to maintain glucose homeostasis. Insulin resistance coupled with insufficient insulin secretion leads to type 2 diabetes. Insulin secretion from pancreatic β-cells is regulated by multiple signaling inputs including the neurotransmitter dopamine. Upon treatment with dopamine, the amplitude and frequency of intracellular free calcium oscillations in β-cells decrease, leading to a concomitant decrease in insulin section. We hypothesize that activation of the dopamine receptor (DRD3) by dopamine releases the Gβγ complex to stimulate a G protein-coupled inwardly-rectifying potassium channel (GIRK3). This inward current keeps the cell polarized, and reduces currents through voltage-gated calcium channels (CaV1.2). This model predicts numerous protein interactions that may only be transient in nature, in particular, we expect close interactions between the DRD3 and GIRK3 as well as each of these with the Gβγ subunit. To test this hypothesis, we are utilizing fluorescence fluctuation spectroscopy to determine protein interactions in stable β-cell lines. Fluorescence fluctuation spectroscopy provides a method to determine interaction between two proteins in a live cell that surpasses many of the limitations of FRET. Single and two color studies allow us to determine diffusion constants of each protein before and after dopamine stimulation and the cross-correlation, or amount of interaction, between two proteins. Additionally, brightness analysis can be used to determine homo- and heterodimerization of the tagged proteins. Diffusion constants, cross-correlation rates, and protein clustering among the DRD3, GIRK3, and Gβγ subunit will be presented. Through this analysis, the dopamine signaling pathway in pancreatic β-cells can be determined, which will reveal novel potential therapeutic targets that can increase insulin secretion during diabetes mellitus.