Development of a robust GABAB calcium signaling cell line using β-lactamase technology and sorting

The GABAB receptor is a member of the "family 3" G protein coupled receptors. The GABAB receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABAB receptors require heterodimerization between two subunits, GABAB₁ and GABAB₂, for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABAB₍₁b₎ and human truncated GABAB₍₂₎ receptors. The cell line was analyzed and sorted using β-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high β-lactamase expression. The single cell clones were further tested in a 384-well calcium mobilization assay using the Fluo-4 AM calcium indicator on the fluorescent imaging plate reader system (FLIPR). Twenty-seven clones were grown up from single cell collections and 10 clones demonstrated a high response to GABA stimulation. The 10 clones were re-evaluated based on agonist dose response and EC₅₀. Clone-16 was identified and utilized in high throughput screening (HTS) assay development. Using sorting and β-lactamase as a reporter, we were able to develop a robust, functional cell-based, GABAB, calcium mobilization assay. The cell line described here can be used for high throughput FLIPR screening and also to compare and rank the potency and selectivity of agonists, antagonists and potentiators of the GABAB receptor. © 2008 International Society for Advancement of Cytometry