Jong Hyun Lim†, Eun Hae Oh‡, Juhun Park§, Seunghun Hong*§⊥, and Tai Hyun Park*†‡∥
†School of Chemical and Biological Engineering, ‡Interdisciplinary Program for Bioengineering, §Department of Physics and Astronomy, and ⊥Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-744, Republic of Korea
∥ Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do 443-270, Republic of Korea
*Address correspondence to Seunghun Hong,Tai Hyun Park
A simple but efficient measurement platform based on ion-channel-coupled receptors and nanovesicles was developed for monitoring the real-time activity of G-protein-coupled receptors (GPCRs). In this work, an olfactory receptor (OR), the most common class A GPCR, was covalently fused with a Kir6.2 channel so that the GPCR action directly induced the opening of the ion channels and changes in the electrical membrane potential without complex cellular signaling processes. This strategy reduced the measurement errors caused by instability of various cellular components. In addition, rather than using whole cells, a cell-surface-derived nanovesicle was used to preserve the membrane-integrated structure of GPCRs and to exclude case-dependent cellular conditions. Another merit of using the nanovesicle is that nanovesicles can be easily combined with nanomaterial-based field-effect transistors (FETs) to build a sensitive and stable measurement platform to monitor GPCR activities with high sensitivity in real-time. Using a platform based on carbon nanotube FETs and nanovesicles carrying Kir6.2-channel-coupled ORs, we monitored the real-time response of ORs to their ligand molecules. Significantly, since this platform does not rely on rather unstable cell signaling pathways, our platform could be utilized for a rather long time period without losing its functionality. This system can be utilized extensively for simple and sensitive analysis of the activities of various GPCRs and should enable various academic and practical applications.
Keywords: ion-channel-coupled receptor; nanovesicle; carbon nanotube; Kir6.2; G-protein-coupled receptor