Development of a robust GABA B calcium signaling cell line using Β-lactamase technology and sorting

Abstract

The GABA B receptor is a member of the “family 3” G protein coupled receptors. The GABA B receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABA B receptors require heterodimerization between two subunits, GABA B1 and GABA B2 , for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABA B(1b) and human truncated GABA B(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 EC 50 . 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, GABA B , 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 GABA B receptor. © 2008 International Society for Advancement of Cytometry