title: New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.
abstract: Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels.
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan. Electronic address: yamamura@phar.nagoya-cu.ac.jp.
authors.forename: Hisao
authors.initials: H
authors.lastname: Yamamura
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Imaizumi
mesh_terms: D001703:Biophysics / Q000379:methods*; D020013:Calcium Signaling / Q000235:genetics*; D002462:Cell Membrane / Q000648:ultrastructure; D055808:Drug Discovery; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D007473:Ion Channels / Q000502:physiology* / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D057054:Molecular Imaging / Q000379:methods*
chemical_list: D007473:Ion Channels
keywords: FRET; Ion channel; Single-molecule imaging; Subunit stoichiometry; TIRF imaging