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Peng Tu

Study of diacylglycerol-activated TRPC6 channels in cortical neurons and their roles in the transport of transition metals



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Published on 23 September 2009


Thesis presented September 23, 2009

Abstract:
Transient Receptor Potential Canonical 6 (TRPC6) channels are diacylglycerol (DAG)-activated non-selective cation channels expressed in a variety of tissues and cells. They are for instance expressed in the cortex of embryonic (E13) mice. Cellular calcium imaging experiments show the existence of DAG-sensitive plasma membrane cation channels in cortical neurons kept in culture. These channels are permeable to Ca2+, Na+, Ba2+ and Mn2+. The Ca2+ entry, not controlled by protein kinase C, is blocked by Gd3+ and SKF-96365 but potentiated by flufenamic acid. Hyperforin, which activates TRPC6 channels without acting on the other TRPC isoforms, triggers an entry of Ca2+ via non-selective cation channels. Although the exact molecular identity of the DAG-sensitive channels in cortical neurons has not yet been established, we suggest that TRPC6 form these channels.
Quantitative analyses with Inductively Coupled Plasma-Optical Emission Spectrometry, atomic absorption spectrometry and synchrotron microbeam X-ray fluorescence (µ-SXRF) show that over-expressing TRPC6 in HEK-293 cells elevates the intracellular contents of zinc, sulphur and manganese. Cellular iron and zinc imaging experiments indicate that TRPC6 channels, either over-expressed in HEK cells or endogenously present in cortical neurons, are permeable to iron and zinc when activated by DAG or hyperforin. The experiments with µ-SXRF further reveal that activating TRPC6 channels in the presence of iron leads to an intracellular iron accumulation in both HEK cells and cortical neurons.
In addition, during the time course of this study, we provided experimental evidence showing that flufenamic acid and hyperforin, two pharmacological tools used to manipulate the activity of TRPC6 channels, release Ca2+ and Zn2+ from mitochondria.

Keywords:
Cortical neurons, HEK-293 cells, TRPC6, diacylglycerol, flufenamic acid, hyperforin, synchrotron microbeam​ X-ray fluorescence, iron, zinc, manganese, copper

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