Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye

Area of Science:

• Pharmacology
• Neuroscience
• Biochemistry

Background:

• High-throughput screening (HTS) is crucial for identifying novel opioid analgesics.
• Existing HTS methods for opioid receptor activation lack naturalistic receptor-effector coupling.
• Development of new pain therapeutics necessitates efficient screening assays.

Purpose of the Study:

• To develop and validate two simple, real-time fluorescence-based assays for μ-opioid receptor (MOPr) activation.
• To enable scalable HTS for potential opioid analgesic compounds.
• To provide assays reflecting naturalistic MOPr signaling.

Main Methods:

• Utilized AtT-20 cells expressing MOPr to measure G protein-gated inwardly rectifying K+ (GIRK) channel activation via membrane potential changes.
• Employed Chinese hamster ovary (CHO) cells expressing MOPr to assess forskolin-induced adenylyl cyclase inhibition via membrane potential.
• Measured membrane potential changes using a proprietary membrane potential-sensitive dye in real-time.

Main Results:

• Demonstrated two distinct, real-time fluorescence assays for MOPr activation.
• Assays successfully measured membrane potential changes indicative of receptor activity.
• The developed assays reflect naturalistic coupling of MOPr to downstream signaling pathways.

Conclusions:

• These fluorescence-based assays offer a scalable and naturalistic approach for HTS of MOPr modulators.
• The assays facilitate the identification of novel opioid analgesics.
• This method advances the development of improved pain management therapeutics.