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Transmembrane transport and storage of zinc in neurons. Roles of TRPC6 channels



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Published on 17 May 2019
Alexandre Bouron, CNRS
Laurence Macari, CNRS

Scientific project

Our current research is focused on the following themes:
- understanding the functions and properties of TRPC6 channels in neurons of the central nervous system;
- Precising the mechanisms by which neurons acquire, store and release Zinc ions.

To address these issues, we mainly use electrophysiological approaches (whole -cell patch-clamp), live-cell fluorescence imaging techniques (with Ca and Zn probes), and molecular biology. Most of the experiments are carried out on cortical neurons kept in primary cultures and isolated from E13 embryonic mice (C57Bl6/J). We also prepare isolated brain mitochondria from neonatal mice to study the release of cations from these organelles.

TRPC6 channels are plasma membrane cation channels allowing the transport of Ca and Na ions. We recently showed that they can also transport Zn ions. The entry of Zn through TRPC6 channels up-regulates the size of the intracellular pools of mobilisable Zn in neurons. Furthermore, over-expressing TRPC6 channels in HEK cells increases their Zn content [Gibon et al., 2011b].

Another key aspect of our activity concerns hyperforin. It is currently known to be one of the main bioactive compounds that underlie the antidepressant actions of the medicinal plant Hypericum perforatum (St John's wort). Of interest, this molecule has the property to enhance the activity of TRPC6 channels. We currently use hyperforin to trigger their opening [Tu et al., 2009]. We could show that, beside its effect on TRPC6 channels, acutely applied, hyperforin releases Zn and Ca from mitochondria [Tu et al., 2010]. Hyperforin is now used to regulate the intracellular concentration of Zn in brain cells [Gibon et al., 2011a].

Experiments are in progress to advance our understanding of hyperforin's mechanisms of action on brain cells. In particular, a special attention is given to its impact on the BDNF receptor TrkB and on adult hippocampal neurogenesis since they appear central in the molecular pathophysiology of depression and in the pharmacological mechanisms of action of antidepressants. We also try to precise its impact on the neuronal homeostasis of Zn.

Biological models, techniques and equipment
We use patch-clamp and imaging methods on living cells (HEK cells, cortical neurons in primary cultures) and isolated mitochondria. The lab is equipped with a patch-clamp setup consisting of an inverted Olympus IX 51 microscope + an Axoclamp 200B amplifier (Axon Instruments) + a motorized micromanipulator (Luigs & Neumann) + a pipette puller (DMZ Universal Puller, Zeitz Instruments).

This patch clamp setup is driven by the Axoclamp software. The lab also possesses a live-cell imaging setup with an inverted Carl Zeiss Axio Observer A1 microscope equipped with a Fluar 40x oil immersion objective lens (1.3 NA) (Carl Zeiss) + the DG-4 wavelength switcher (Princeton Instruments) + a cooled CCD camera (CoolSnap HQ2, Princeton Instruments). Images are acquired and analyzed by means of the MetaFluor (Universal Imaging) software.