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Metalloneurochemistry is a subfield of bioinorganic chemistry, which studies the functions of metal ions in the brain and nervous system at the molecular level. Although metal ions and other inorganic species have numerous vital functions in the central nervous system, the interest of bioinorganic chemists in this area was initially minimal because many of the challenges in the field involve studying spectroscopically silent metal ions like Ca2+, Mg2+, Zn2+, K+, and Na+. Investigating problems in neurochemistry may be less appealing to bioinorganic chemists because of the inability to apply conventional instrumental techniques, such as EPR, Mössbauer, UV-visible, and extended x-ray absorption fine structure (EXAFS) spectroscopy, to analyze such metal ions. As a result, biologically compatible and non-intrusive techniques have been developed, foremost among them fluorescence microscopy imaging with molecular probes. Recently, the field has evolved to include the study of the biological functions and mechanisms of action of small signalling molecules like nitric oxide (NO), nitroxyl (HNO), and hydrogen sulfide (H2S). The following sections highlight a few topics in metalloneurochemistry.

The Structure and Function of Ion Channels
The two main classes of ion channel proteins are ligand-gated and voltage-gated. Voltage-gated channels, like the potassium crystallographically-sited activation channel (KcsA) channel, open and close in response to changes in membrane potential. Ligand-gated channels, like the MthK K+ channel, require the binding of an external substrate to open the channel. The MthK K+ channel opens and closes in a Ca2+-dependent fashion, and crystallographic analysis reveals two calcium-binding sites on the intracellular side of the protein. Free energy supplied by Ca2+-binding induces structural changes that open and close the pore. The elucidation of the x-ray crystal structure of the bacterial KcsA K+ channel, was a landmark in the study of ion channel metalloneurochemistry. The rapid propagation of neurochemical signals requires the K+ channel to discriminate against Na+ ions and to allow passage of K+ ions at a rate of 107 to 108 s-1. The potassium channel is a 4-fold symmetric tetramer resembling a teepee that surrounds a central pore. Each of the four subunits of the channel has a sequence of five amino acids, Thr-Val-Gly-Tyr-Gly, on the extracellular side of the channel that comprises the selectivity filter. The carbonyl oxygen atoms of these five amino acid residues are oriented in a configuration capable of coordinating completely dehydrated K+, but not the smaller Na+ ions. Electrostatic repulsion between K+ ions, which pass through the selectivity filter pore in single file, provides the force necessary to drive the ions through the channel at a high rate of speed.

Calcium Proteins and Synaptic Transmission
Perhaps the most important metal ion in signal transduction is Ca2+. Changes in intracellular Ca2+ concentrations initiate a cascade of signaling events. Calmodulin, an intracellular protein capable of coordinating four Ca2+ ions, binds to a wide variety of enzyme targets, modulating their states of activation. Evidence now exists that Ca2+-dependent translocation of calmodulin in neuronal nuclei is critical both for rapid signaling and for memory formation. Calcineurin, which was first isolated from mammalian brain, is one target of calmodulin involved in Ca2+- dependent cellular signaling. Another family of Ca2+-containing proteins involved in the release of neurotransmitters was identified known as synaptotagmins. Neurotoxicity of heavy metals such as lead may arise from competitive binding to the Ca2+ locale in the C2 domains of the synaptotagmin. Competitive metal binding studies indicate that the affinity of synaptotagmin for Pb2+ is significantly higher than for Ca2+. Selective chelators for Pb2+ removal may provide a mean to overcome this heavy metal neurotoxicity.

Calcium Sensing and Imaging
The spectroscopically silent nature of Ca2+ has prevented for a long time its detection and tracking in biology. Calcium imaging has evolved as a technique designed to sense Ca2+ selectively over other metal ions and signaling agent, and takes advantage of fluorescent molecules that can respond to the binding of Ca2+ ions by changing their fluorescence emission properties. Two main classes of calcium indicators (sensors) exist: small-molecule probes and genetically encoded proteins.

Small-molecule calcium sensors are based on an analogue of EGTA called BAPTA, with high selectivity for calcium (Ca2+) ions versus magnesium (Mg2+) ions. Genetically encoded proteins are fluorescent proteins derived from the green fluorescent protein (GFP) or its variants, fused with calmodulin (CaM) and the M13 domain of the myosin light chain kinase, which is able to bind CaM. Alternatively, variants of GFP are fused with the calcium binding protein troponin C (TnC), applying the mechanism of FRET (Förster Resonance Energy Transfer) for signal modulation. Genetically encoded indicators do not need to be loaded onto cells, instead the genes encoding for these proteins can be easily transfected to cell lines. The discovery, expression, and development of GFP has revolutionized the fields of fluorescence imaging and neurobiology, culminating with the award of the 2008 Nobel Prize in Chemistry to Roger Tsien, Martin Chalfie and Osamu Shimomura.

Calcium imaging can be used to optically probe intracellular calcium in living animals. This technique has allowed studies of neuronal activity in hundreds of neurons and glial cells within neuronal circuits.

Metallochaperones and Metal Ion Homeostasis
Many neurological disorders have been associated with the accumulation of toxic amounts of metal ions in the nervous system. The seminal work on the iron-transport protein transferrin and the iron-storage protein ferritin epitomizes the importance of tight regulation of metal ion concentrations. Other examples include the copper chaperone for superoxide dismutase (SOD1), which facilitates delivery of copper ions to the enzyme.

Zinc transporters and Synaptic Zn2+
Zinc (Zn2+) is the second most abundant transition metal in the body, after iron. Even though most divalent zinc is tightly bound to metalloproteins, pools of loosely-bound, “mobile” Zn2+ are found in specialized, secretory tissues such as the prostate, pancreas, and brain. Of special interest is the role of mobile Zn2+ in the nervous system, where high concentrations of occur in specific regions of the brain. Mobile Zn2+ is primarily restricted to the forebrain, where zinc-containing axons are particularly abundant in the hippocampus, cortex, olfactory bulb, and amygdala. The function of synaptic Zn2+ has been the subject of much research. High concentrations of Zn2+ in the olfactory bulb of mice and the occurrence of two kinds of zinc-enriched terminals suggest a role in olfaction. Zinc may bind to receptors on the postsynaptic neuron and modulate the flow of ions through channels, or it may act as an intracellular signaling agent following entry into the postsynaptic neuron through Zn2+-permeable channels. When mice with the zinc transporter ZnT3 protein knocked out are crossed with transgenic mice expressing a mutant human amyloid precursor protein, a significant reduction was observed in the formation of β-amyloid plaques that are commonly found in Alzheimer’s patients.

Metallothioneins and Zn2+
Metallothioneins are cysteine-rich proteins having a number of functions in the central nervous system, including heavy-metal detoxification. MT-III exists at lower levels in the brains of Alzheimer’s patients, although Alzheimer’s disease neuronal changes may not be a consequence of such down-regulation. There is also suggestion that MT-III may be responsible for converting NO signals into Zn2+ signals.

Fluorescent Sensors for Biological Metal Ions and Reactive Nitrogen and Oxygen Species
Studying spectroscopically silent metal ions like Zn2+ and small inorganic radicals like NO require methodologies to visualize these otherwise undetectable species in neuronal cells. The first generation of fluorescent probes available for Zn2+ were quinoline sulfonamides such as TSQ and Zinquin, even though their fluorescence properties are not ideal for application to metalloneurochemistry. Later generations of Zn2+ sensors including Zinpyr 1 (ZP1), ZP2 , ZP4 , and Rhodafluor 2 (RF2) are more amenable to biological investigations. Related sensors for detection of biological nitric oxide have also been reported. The first small-molecule fluorescent probes for NO relied on an indirect detection through the reaction of an oxidized product of NO (NO2 or N2O3) with o-diaminobenzene analogues, as in the DAF family of probes. In 2006, the first direct sensor for biological NO, CuFL1, was created. CuFL1 is a Cu2+-based, NO-specific probe containing a fluorescent ligand which is nitrosated upon addition of NO, a process which releases the copper ion and leads to bright fluorescence emission in the FL1-NO product. Fluorescent small molecule sensors for nitroxyl (HNO), the one-electron reduced sibling of NO, were also developed, as were probes for Cu2+, H2S, biological thiols, and other reactive oxygen and nitrogen species present in cells.