Hot off the Press

Hot off the Press highlights recently published work for the benefit of our readers. Our contributor this month has focused on antidepressant inhibition in neurotransmitter transporters. New contributors are always welcome. If you are interested please contact molbiosyst@rsc.org for more information, we’d like to hear from you.

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Molecular view of antidepressant inhibition in neurotransmitter transporters

Reviewed by Kutti R. Vinothkumar, MRC-LMB, Cambridge, UK.

 

Cell to cell communication between nerve cells of the central nervous system is mediated by the release of neurotransmitters at the synaptic junctions where they activate the postsynaptic ion-channels and receptors. The precise control of the intensity and the lifetime of these released neurotransmitters are critical for many different cellular processes. The released neurotransmitters can either be cleaved by enzymes or transported back to presynaptic nerve cells to replenish the synaptic vesicles by transporters.

The transmembrane (TM) proteins involved in the reuptake of neurotransmitters are classified as the neurotransmitter sodium symporter family (NSS). Malfunction of these transporters have been implicated in human disorders such as depression, epilepsy and Parkinson's disease. Not only are they drug targets for anticonvulsants and antidepressants but also targets for addictive substances such as cocaine. Tricyclic (TCA) antidepressants are non-competitive inhibitor of the NSS family with profound pharmacological interest.

The structure of LeuT, a bacterial homologue of the NSS family provided the first glimpse of the architecture of these medically important transporters. As expected the protein core had 12 TM domains with substrates leucine and sodium bound halfway across the membrane bilayer, in a site devoid of water. Partially unwound TM helices, with their exposed main-chain atoms and helix dipoles play a major role in substrate binding. The structure with substrates bound represents the occluded state where both the extracellular and intracellular gates are closed. Conformational changes in TM helices have been proposed to occur during the uptake and release of substrates. One obvious question that is asked frequently is if the TCA antidepressants inhibit also LeuT, which would help to understand how these inhibitors prevent the reuptake of neurotransmitters at the syanpse.

Starting with a screen of wide range of inhibitors, the groups of Eric Gouaux and Da Neng Wang independently provide biochemical and structural evidence that LeuT is indeed inhibited by TCA antidepressants. The crystal structures show that the inhibitors bind in a vestibule facing the extracellular side, 11 Å above the substrate-binding site providing a molecular view of non-competitive inhibition in a transporter. Binding of inhibitor stabilizes the extracellular gate in a closed conformation there by preventing the release of substrate.

Using the structure of LeuT as the model, Zhou et al. have gone a step further by mutating key residues involved in TCA binding sites of the human neurotransmitter transporters which resulted in decrease efficiency of inhibition, clearly indicating that the TCA-binding site is probably conserved in both in prokaryotic and eukaryotic NSS proteins. These studies open up new path in designing specific and efficient drugs for the neurotransmitter transporters there by preventing the reuptake of neurotransmitters from the synapse.

 

Zhou Z., Zhen J., Karpowich N. K., Goetz R. M., Law C. J., Reith M. E. A., & Wang D. N., Science, 2007, 317, 1390–3.

Singh S. K., Yamashita A., & Gouaux E., Nature, 2007, 448, 952–6.

Hot off the RSC Press

A perfect partner for DNA extraction

Reviewed by: Wendy Crocker, Royal Society of Chemistry, Cambridge, UK.

 

‘Mass-production of DNA and RNA in industry’ is the future offered by researchers at Kyushu University, Fukuoka, Japan, who are using modified DNA molecules to catch and contain specific sequences of DNA. The DNA molecules in this pure form can be used to identify genetic problems that cause disease and in the future could be used as drugs.

Single-stranded DNA continually searches for a matching strand to pair up with, in a process known as hybridisation. A DNA strand can only hybridise properly with another strand that is its perfect partner. To find and separate specific DNA sequences Tatsuo Maruyama and colleagues attached the DNA partner of a specific DNA sequence to a hydrophobic tail. This formed what the researchers called a DNA-surfactant.

Surfactants have distinct hydrophilic and hydrophobic sections. When surfactants are extracted into oil they form clusters called reverse micelles, which are spherical structures that hide their hydrophilic heads in the centre of a hydrophobic shell.

The Japanese researchers found that DNA molecules that hybridised with the DNA-surfactant became part of the hydrophilic core of reverse micelles and so could be extracted into oil. DNA molecules that were not perfect partners to the DNA-surfactant weren’t hybridised and were left behind, along with any other hydrophilic biomolecules that might otherwise have contaminated the DNA.

Paschalis Alexandridis, a surfactant expert at the State University of New York at Buffalo said, ‘I am very pleased to see the recognition capabilities of DNA being applied to the practical – and scaleable – setting of reverse micellar extraction.’

Maruyama’s team say the potential for mass production of high purity DNA is ‘of great importance’. The team foresees that their procedure will help reduce the cost of these highly-specific DNA molecules, which are expected to find use as analytical tools and drugs in the near future.

 

T. Maruyama, T. Hosogi and M. Goto, Chem. Commun., 2007, DOI: 10.1039/b708082d.

Cell preservation all wrapped up

Reviewed by: Kathleen Too, Royal Society of Chemistry, Cambridge, UK.

 

Freezing cells inside glass cages could potentially improve human fertility treatments.

Utkan Demirci and Grace Montesano, at Harvard Medical School and the Massachusetts Institute of Technology, US, have developed the first high-throughput cell vitrification method for automated cell preservation. Demirci and Montesano’s research involves cell encapsulation in droplets; ‘the aim is to apply the technology to real problems in medicine,’ said Demirci.

Demirci and Montesano’s cell preservation method works by trapping single cells in droplets of a cryoprotectant—a liquid that prevents cell damage on freezing—and the droplets are then vitrified. Vitrification is a rapid freezing process in which a fluid turns into a glass-like solid without crystal formation. The new procedure can preserve cells at rates as high as thousands cells per second while retaining cell viability. It also allows lower concentrations of toxic cryoprotectant such as 1,2-propanediol to be used, leading to significantly reduced osmotic stress on the cells. Furthermore, automation avoids human error and minimises mechanical stress to the cells due to manual handling. Demirci and Montesano’s cell preservation method works by trapping single cells in droplets of a cryoprotectant—a liquid that prevents cell damage on freezing—and the droplets are then vitrified. Vitrification is a rapid freezing process in which a fluid turns into a glass-like solid without crystal formation. The new procedure can preserve cells at rates as high as thousands cells per second while retaining cell viability (Fig. 1). It also allows lower concentrations of toxic cryoprotectant such as 1,2-propanediol to be used, leading to significantly reduced osmotic stress on the cells. Furthermore, automation avoids human error and minimises mechanical stress to the cells due to manual handling.

Fig. 1 Vitrified cells (left) can be thawed (right) and remain viable.

Among the different cells preserved were liver cells and mouse embryonic stem cells and Demirci suggests that future work could provide controlled vitrification methods for reproductive (germ) cell preservation. ‘This could have impact in extending human fertility, allowing higher yields and success,’ said Demirci. ‘One challenge in vitrifying germ cells is their larger size compared to other cell types. We will optimise our system to address challenges in this arena by changing the droplet sizes and concentrations.’

David Juncker, an expert in high-throughput cell analysis from McGill University, in Montreal, Canada, explained that ‘cell preservation and manipulation is of great interest. The method seems versatile,’ he added, ‘I could imagine using it for rare stem cell collection and conservation.’

 

U. Demirci and G. Montesano, Lab Chip, 2007, DOI: 10.1039/b705809h

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