Fluorescence-based method for analysis of glycine receptor alpha 3 agonists

Glycine, a key neurotransmitter, plays a complex role in the central nervous system (CNS). It acts as an inhibitory neurotransmitter by interacting with ligand-gated chloride channels. Glycine plays a crucial role in pain and itch transmission through its interactions with the glycine receptor alpha 1 (Glyr α1) and 3 (Glyr α3) subunits. Targeting glycinergic neurotransmission in the CNS, particularly the spinal cord, could provide a beneficial strategy for analgesic and/or abirritant drug discovery. Currently, the search for novel pharmacological agonists and modulators against glycine receptors is still in its early stages.

In this study, we designed two different vectors to express the human hetero-pentameric GlyR α3β. We tested different chemical transfection protocols and performed fluorescence endpoint measurements in acutely transfected cells and cells that underwent antibiotic selection. We further validated our findings by studying the electrophysiological properties of the vector-expressing cells using patch clamp.

The results demonstrate that acutely vector-transfected cells and vector-transfected cells subjected to antibiotic selection work equally well during fluorescence-based measurements. However, in single-cell measurements such as patch clamp, acutely transfected cells perform better. The results further show that reversing the equilibrium potential of [Cl-] can enhance the fluorescence response from the FluoVolt™ membrane potential dye, which can be used during patch clamp measurements. This establishes a simple and cost-effective method to screen for compounds in cell cultures using chemical transfection with a vector expressing GlyR α3β, which could aid in developing the next generation of non-opioid analgesic.