E-Conference: Defining Calcium Entry Signals
TRPC channels and the second messengers that regulate them
12 June 2004
Randen L. Patterson
I only have a few comments:
"I completely agree that as yet there is insufficient experimental evidence around to state that DAG is the physiological second messenger of TRPC3/6/7 gating.
Further milestones in our understanding of TRPC activation would be the identification of DAG binding sites in TRPC channels (if direct binding actually occurs), genetic manipulation of these sites and then testing for in vivo consequences. There is still a long way to go on this path."
1)This is true. Until the molecular biology and protein biochemistry is performed to ascertain whether or not DAG or its analogues can have a direct effect on TRPC channels, all other discussion is merely hypothesis driven, without strong experimental evidence to support it.
"However, if a different receptor were chosen, for instance the endogenous muscarinic receptor in HEK cells or a coexpressed H1 histamine receptor, OAG challenge subsequent to receptor stimulation would not elicit any measurable Sr2+ or Ca2+ influx. Thus, most probably the quantitative aspect of receptor-induced PLC activation determines whether a secondary OAG challenge has an effect or not. "
2) We used the endogenous muscarinic receptor in that figure. These types of explanations are merely handwaving, see 1).
"However, there is nothing wrong with the speculation that "DAG may be the decisive second messenger generated by PLC". In the scientific literature, it is discussed controversially how DAG-sensitive TRPC channels are activated. I am very puzzled by the finding that some investigators claim that TRPC3 stably expressed in HEK293 cells (T3- clones) behaves as a store-operated, Ca2+-permeable channel gated by conformational coupling with the IP3 receptor, while others working with the same cell clone provide evidence that the same ion channel is gated store-independently, subsequent to PLC activation by a mechanism involving DAG. At present, I have no explanation for these discrepant results."
3)As I have stated in previous comments, one of the biggest problems with this field is that very few researchers are looking at endogenous TRPC channels. With the advent of siRNA, there is no excuse that those who use fluorescence OR ELECTROPHYSIOLOGY to specifically measure calcium entry via TRPC channels, do not to delete endogenous TRPC channels from their system. This would allow one to look for specific endogeous effects of TRPC channels, which could then be rescued with overexpression of exogenous TRPC channels, and more importantly TRPC channel mutants.
"It does not come as a surprise that the role of lipid messengers for the activation of TRP and TRPL in the Drosophila eye is also a moot issue. Flies impaired in DAG kinase activity (leading to an increase in the local DAG concentration upon receptor stimulation) show enhanced spontaneous currents and light responses consistent with the concept that DAGs or metabolites, such as polyunsaturated fatty acids (PUFAs), gate the cation channels. An alternative explanation is based on the role of PUFAs as metabolic uncouplers and the observation that metabolic inhibition activates light-sensitive TRP and TRPL channels. However, there is recent evidence that metabolic inhibition primarily impairs DAG kinase activity consistent with the notion of TRP channel gating by DAG."
4)Since TRPC channels have evolved quite significantly from fly, it is unreasonable to think that they would have to retain the same activation mechanisms that are used in fly. As humans have already done away with TRPC2, which was an evolution of TRP from fly, it is my opinion that even when the activation of TRP in fly eye is determined, it will provide little insight into the activation and regulation of mammalian TRPC channels.
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