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E-Conference: Defining Calcium Entry Signals

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TRPC channels and the second messengers that regulate them

8 June 2004

Thomas Gudermann

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.

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.

Honestly, I tend to be a little bit reserved with regard to the conclusions drawn from Figures 2H and 2I of Ma et al., Science 287:1647-1651, 2000. Simply looking at the morphology of cells treated with calyculin A illustrates that many processes are going on in these cells, one of which may be the disruption of TRPC/IP3 receptor contacts. Considering these drastic morphological changes, I personally find distinct functional differences between untreated and treated cells very difficult to interpret. Figure 2H would indeed indicate that receptor and OAG stimulation of TRPC3 are independent phenomena. 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.

This general view is further substantiated if one examines TRPC3 or 6activity electrophysiologically. The pro's and con's of fluorescence imaging versus electrophysiology have already been discussed in detail in other contributions to this E-conference, so I will not elaborate on this issue again. Suffice it to say, conclusions drawn gain credibility if both methods yield compatible results. Along these lines, at least two labs have shown independently by means of electrophysiology that receptor and OAG stimulation of cells have no additive effect on TRPC3 or TRPC6 current amplitude. Therefore, I am not yet convinced that receptor and DAG stimulation of TRPCs rely on independent mechanisms.

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.

Last not least, can we really be sure that exogenously added membrane-permeable DAG analogs like OAG and DOG are not at all different from endogenously produced DAGs like SAG (steaoryl-arachidonoyl-glycerol) or SLG (stearolyl-linoleoyl-glycerol) with regard to their biological effects? How do we know about the local concentration of these DAGs following PLC stimulation? Chemically and biophysically SAG and SLG are clearly distinct from DAG and DOG. Thus, as stated before I would only feel confident with experimental results if external addition of membrane-permeable DAGs yield results compatible with those obtained after pharmacological blockade of DAG lipase and kinase and after addition of membrane-impermeable endogenous DAGs. Needless to say, the best way to study these "natural" DAGs is electrophysiology...

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