RRC ID 64935
Author Hirata T, Terai T, Yamamura H, Shimonishi M, Komatsu T, Hanaoka K, Ueno T, Imaizumi Y, Nagano T, Urano Y.
Title Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.
Journal Anal Chem
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.
Volume 88(5)
Pages 2693-700
Published 2016-3-1
DOI 10.1021/acs.analchem.5b03970
PMID 26894407
MeSH Boron Compounds / chemistry* Cations, Monovalent Cell Membrane / metabolism* Crown Ethers / chemistry* Fluorescent Dyes / chemistry* HEK293 Cells HT29 Cells HeLa Cells Humans Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / metabolism Potassium / metabolism* Potassium Ionophores / pharmacology
IF 6.785
Human and Animal Cells HeLa