Authors:

Olivia Schmidt

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About Paper:

recently, the tools to study this neural circuitry were lacking. Genetically encoded opioid peptide indicators provide the solution to this problem, allowing for measurement of real time opioid receptor activity at the resolution of a single cell. These engineered sensors are optimized versions of the native receptor that have been mutated to contain a chromophore, such as a green fluorescent protein (GFP). Upon binding the appropriate opioid ligand, the sensor undergoes a conformational change that stabilizes the chromophore and causes a measurable fluorescence change. The mu opioid receptor (MOR), which is the subtype of opioid receptor involved in pain regulation and binds drugs like heroin and oxymorphone, has been an exceptionally difficult target for sensor design, with previous MOR sensors having substantial limitations. The Abdelfattah Lab has been working on its own GFP-based MOR sensor, which has significant improvements in membrane localization, basal fluorescence, and fluorescent response. We developed a GFP-based MOR sensor by targeted mutagenesis of amino acid residues in close proximity to the GFP chromophore and the fusion site between the GFP and MOR receptor. We made combination libraries between positions that showed improved response and were able to further improve brightness and response to reach approximately 10-fold change in response upon ligand addition. In addition, we recently started developing a far-red version of the MOR sensor. Overall, far-red sensors have better in vivo applicability, as they allow for imaging deeper in tissue and better image resolution. To make this far-red MOR sensor, we replaced the GFP of the current design with a HaloTag, a self-labeling protein that can bind fluorescent dyes, including far-red dyes. Further development of this sensor will involve iterative additions and deletions in the regions that connect the MOR to the HaloTag and point mutations close to the dye binding pocket, all in order to optimize this 95 MOR-HaloTag sensor for far-red dyes specifically. Ultimately, a functional, red-shifted genetically encoded MOR sensor would be incredibly valuable for understanding opioid neuropeptide circuitry, which is vast and implicates regions deep within the brain. Om Taropawala:

Source:

Brown / SPRINT|Undergraduate Teaching and Research Awards (UTRA)

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Co-authors:

Olivia Schmidt