| Autor: |
Hirata T, Terai T, Yamamura H; Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya 467-8603, Japan., Shimonishi M, Komatsu T, Hanaoka K, Ueno T, Imaizumi Y; Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya 467-8603, Japan., Nagano T, Urano Y |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical chemistry [Anal Chem] 2016 Mar 01; Vol. 88 (5), pp. 2693-700. Date of Electronic Publication: 2016 Feb 19. |
| DOI: |
10.1021/acs.analchem.5b03970 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
