Development of a robust GABA(B) calcium signaling cell line using beta-lactamase technology and sorting.
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 beta-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high beta-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 beta-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.
Cui
Mei
M
Cancer Biology, Pfizer Global Research & Development, 10724 Science Center Drive, San Diego, California 92121, USA.
Chung
Fu-Zon
FZ
Donahue
Christopher J
CJ
D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D009943:Organophosphorus Compounds / Q000494:pharmacology; D018080:Receptors, GABA-B / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism*
D009943:Organophosphorus Compounds; D018080:Receptors, GABA-B; C107353:CGP 54626; D001618:beta-Lactamases