Selective Zinc Sensor Molecules with Various Affinities for Zn²⁺, Revealing Dynamics and Regional Distribution of Synaptically Released Zn²⁺ in Hippocampal Slices

Abstract

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.

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

“…1). ZIMIR consists of three moieties: a fluorophore based on fluorescein, a Zn 2+ binding motif derived from dipicolylamine (17)(18)(19), and a pair of dodecyl alkyl chains for membrane tethering. In the absence of Zn 2+ , the fluorescence of ZIMIR is quenched by the photo-induced electron transfer from the amino group to fluorescein.…”

A new rhodamine B based fluorometric chemodosimeter for Cu2+ ion in aqueous and cellular media

“…The development of highly sensitive, selective, and cell membrane permeable probes that exhibit a visible "turn-on" fluorescent emission in aqueous media is very much necessary for fluorescence imaging of metal ions in living cells. Till date a number of fluorescence probes has been reported for imaging various intracellular heavy metal ions such as Zn 2 þ , Cd 2 þ , Cu þ , Pb 2 þ , Fe 3 þ , Hg 2 þ and Cu 2 þ [14][15][16][17][18][19][20].…”

Mechanism and Regulation of Cellular Zinc Transport

“…Powerful tools to address these issues, e.g., fluorescent zinc sensors targeted to specific organelles, have been developed and are being constantly refined (65)(66)(67)(68)(69). The advantage of such an approach has been demonstrated previously for H + and Ca 2+ using GFP-based chameleon and pHluorin constructs.…”