36 - Expression of Ion Channel-Coupled Receptors using Human Coupled IVTT Cell-Free Expression
University of Arizona
G-protein-coupled receptors (GPCRs) transduce extracellular stimuli via ligand binding into intracellular signals. Fusion of a GPCR to an ion channel yields chimeric proteins, referred to as ion channel-coupled receptors (ICCRs),which directly transduce ligand binding into an electrical signal in the absence of downstream signaling. ICCRs couple ligand-induced GPCR conformational changes to ion channel activity; thereby converting chemical signals into readily measurable electrical current. This paradigm extends the detectable, chemical space of electrophysiological measurement techniques to GPCR ligands, enabling advancements in chemical sensing and drug-screening applications. We investigated the use of a human coupled in vitro transcription-translation (IVTT) expression system to produce four proteins, a fluorescent marker (EGFP), an ion channel (EGFP-Kir6.2?26), a GPCR (glucagon receptor), and an ICCR (human dopamine D2-Kir6.2). These constructs have successfully been cloned into the IVTT expression vector, pT7CFE1-CGST-HA-His. Preliminary results show that EGFP-Kir6.2?26 has been successfully expressed using the human coupled IVTT expression system; however, continued work aims to optimize its expression. This work is funded by the Beckman Foundation and the NIH.