A new mitochondrial fluorescent zinc sensor

doi:10.1016/S0143-4160(03)00122-2

Cell Calcium

Volume 34, Issue 3, September 2003, Pages 281-284

https://doi.org/10.1016/S0143-4160(03)00122-2 Get rights and content

Abstract

A novel cationic fluorescent zinc (Zn²⁺) indicator (RhodZin-3) with nanomolar affinity for Zn²⁺ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca²⁺ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn²⁺ ([Zn²⁺]_m).

Introduction

The zinc(II) ion (Zn²⁺) has been long known to play critical roles in protein structure and function [1]. Recently Zn²⁺ has emerged as an important player in neurotransmission [2] and neuronal injury [3], [4]. Much of the total biological zinc is tightly bound to proteins and enzymes [5]. Rapid rises in intracellular free Zn²⁺ ([Zn²⁺]_i) have been linked to neuronal injury in transient global ischemia and epilepsy [2], [4]. The mechanisms by which Zn²⁺ exerts potent neurotoxic effects are still largely unknown. We and others have suggested that among the intracellular targets of Zn²⁺ dependent neurotoxicity, Zn²⁺ sequestration into mitochondria may play a critical role [6], [7], [8]. As with Ca²⁺, upon excessive cytosolic Zn²⁺ loading, mitochondria take up Zn²⁺ and help to restore intracellular Zn²⁺ homeostasis [8]. However, once in the mitochondria, Zn²⁺ can trigger a prolonged disruption of the functioning of these organelles. Indeed, Zn²⁺ has been shown to have potent effects on mitochondria [7], [8], [9], [10], [11], [12], [13], [14]. For instance, in neuronal mitochondria, rises in [Zn²⁺]_m promote loss of mitochondrial membrane potential (ΔΨ_m) and generation of reactive oxygen species (ROS) as well as release of pro-apoptotic factors [7], [8], [14], [15].

Direct measurements of [Zn²⁺]_m have previously exploited the Zn²⁺ sensitivity of rhod-2, a mitochondrial probe usually employed to detect mitochondrial Ca²⁺ uptake [15]. However, the high Ca²⁺ sensitivity of rhod-2 potentially confounds such zinc measurements. Thus, better tools are critically needed to effectively explore changes in [Zn²⁺]_m. Herein we described a novel fluorogenic zinc-specific sensor that localizes into mitochondria of cultured neurons and can effectively detect rises in [Zn²⁺]_m.

Section snippets

Materials

Reagents were obtained from Sigma Chemical Company (St. Louis, MO, USA) or Aldrich Chemical Company (Milwaukee, WI, USA) and used as received. Probe synthesis was performed at Molecular Probes, Inc.

In vitro characterization of RhodZin-3

Absorbance and emission spectra, dissociation constants, and fluorescence enhancements were measured in standard fashion [16]. Spectra were measured at 22 °C in 100 mM KCl, 50 mM MOPS, pH 7.0. Free [Zn²⁺] in buffered solutions was determined using WEBMAXC v2.10. Free Zn²⁺ solutions of 0.7, 2.75, and 11

Results and discussion

Condensation of the aldehyde 1 [18] with two equivalents of 3-dimethylaminophenol afforded the unstable dihydroxanthene 2, which was quickly oxidized with p-chloranil to give the xanthene 3 (Scheme 1). The methyl esters were removed by saponification, and the resulting salt form of RhodZin-3 (4) converted into its cell permeable AM ester [19] form 5 by acidification and reaction with bromomethyl acetate. RhodZin-3 utilizes the cationic rhod fluorophore [20], coupled to the N,N,N′-triacetic acid

Acknowledgements

K.R.G. and A.R. thank Dr. Iain Johnson for helpful discussions, and Hans Engel for expert technical assistance. We also thank Simin Amindari for expert assistance with the cell cultures. This work was supported by NIH grants NS30884 and AG00836 (JHW), AG00919 (SLS), and a grant from the Alzheimer’s Association (JHW).

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Synaptic Zn²⁺ plays an important role in neurotransmission and a neuromodulator. The development of the imaging tools for monitoring spatiotemporal changes taking place in synaptic Zn²⁺ concentrations is necessary in order to understand the role of Zn²⁺ in the function of many aspects of the glutamate system. In this work, two-photon probes 1 and 2, bearing ifenprodil-like tails that have affinity for NMDA receptors of neuronal cells, were designed and prepared. The two-photon fluorescent probe 1, which bears (N-(6-acetylnaphthalen-2-yl)-N-methylglycine) as two-photon fluorophore, enables high resolution imaging of neuronal cells. The two-photon fluorescent probe 2, which contains the di-2-picolylamine (DPA) as a Zn²⁺-binding site, the naphthalimide unit as the two-photon fluorophore, and the ifenprodil-like tail as the NMDA receptor binding moiety, can be employed for selective detection of Zn²⁺ located near the NMDA receptor and for monitoring concentration changes of Zn²⁺ in live neurons and hippocampal tissues.

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A new mitochondrial fluorescent zinc sensor

Abstract

Introduction

Section snippets

Materials

In vitro characterization of RhodZin-3

Results and discussion

Acknowledgements

Int. Rev. Neurobiol.

Exp. Neurol.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Neurobiol. Dis.

J. Biol. Chem.

Cell Calcium