Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices.
We have developed a series of fluorescent Zn(2+) sensor molecules with distinct affinities for Zn(2+), because biological Zn(2+) concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have K(d) values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn(2+) within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn(2+) concentration, while that of ZnAF-3 (K(d) = 0.79 muM) was not saturated even at relatively high Zn(2+) concentrations. In hippocampal slices, we measured synaptic release of Zn(2+) in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn(2+) concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn(2+) concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn(2+) in biological applications.
Komatsu
Kensuke
K
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kikuchi
Kazuya
K
Kojima
Hirotatsu
H
Urano
Yasuteru
Y
Nagano
Tetsuo
T
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D016466:CHO Cells; D002413:Cations, Divalent; D002614:Chelating Agents / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D005029:Ethylenediamines / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
D002413:Cations, Divalent; D002614:Chelating Agents; D005029:Ethylenediamines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine