title: Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.
abstract: K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hirata
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya 467-8603, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Yamamura
authors.forename: Manabu
authors.initials: M
authors.lastname: Shimonishi
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya 467-8603, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Imaizumi
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D001896:Boron Compounds / Q000737:chemistry*; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000378:metabolism*; D043844:Crown Ethers / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D061216:Potassium Ionophores / Q000494:pharmacology
chemical_list: D001896:Boron Compounds; D002414:Cations, Monovalent; D043844:Crown Ethers; D005456:Fluorescent Dyes; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D061216:Potassium Ionophores; C000621153:TLSHalo compound; D011188:Potassium