Coupling of olfactory receptor and ion channel for rapid and sensitive visualization of odorant response.
In the human smell sensing system, there are about 390 kinds of olfactory receptors (ORs) which bind to various odorants with different affinities and specificities. Characterization and odorant binding pattern analysis of the ORs are essential for understanding of human olfaction and to mimic the olfactory system in various applications. Although various cell-based odorant screening systems have been developed for this purpose, many human ORs (hORs) still remain orphan because of the time-consuming and labor-intensive experimental procedures of the available screening methods. In this study, we constructed an ion channel-coupled hOR for simple odorant detection by rapidly visualizing the odorant response to overcome the limitations of conventional screening systems. The hORs were coupled to the Kir6.2 potassium channel and the fusion proteins were expressed in HEK293 cells. In this system, when an odorant binds to the hORs coupled to the ion channel, a conformational change in the OR occurs, which consequently opens the ion channel to result in ion influx into the cell. This ion influx was then visualized using a membrane potential dye. Cells expressing ion channel-coupled hORs showed high sensitivity and selectivity to their specific odorants, and the odorant-hOR binding pattern was visualized to identify the response of individual hORs to various odorants, as well as the response of various hORs to various odorants. These results indicate that the ion channel-coupled hOR system can be effectively used not only for simple and fast high-throughput odorant screening, but also to visualize the odorant-hOR response pattern.
Interdisciplinary Program of Bioengineering, Seoul National University, Seoul 151-742, Republic of Korea.
Eun Hae
EH
Oh
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Seung Hwan
SH
Lee
Bio-MAX Institute, Seoul National University, Seoul 151-742, Republic of Korea.
Hwi Jin
HJ
Ko
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Jong Hyun
JH
Lim
Interdisciplinary Program of Bioengineering, Seoul National University, Seoul 151-742, Republic of Korea; School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea; Bio-MAX Institute, Seoul National University, Seoul 151-742, Republic of Korea; Advanced Institutes of Convergence Technology, Suwon 443-270, Republic of Korea. Electronic address: thpark@snu.ac.kr.
Tai Hyun
TH
Park
D015153:Blotting, Western; D003001:Cloning, Molecular; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D009812:Odorants; D011188:Potassium / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D018035:Receptors, Odorant / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000099161:Kcnj11 Channel
D011188:Potassium; D024661:Potassium Channels, Inwardly Rectifying; D018035:Receptors, Odorant; D000099161:Kcnj11 Channel
Ion channel-coupled hOR; Membrane potential dye; Odorant detection; Odorant response pattern