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E-Conference: Defining Calcium Entry Signals

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TRPC channels and the second messengers that regulate them

21 June 2004

Raghu Padinjat

As Randen Patterson has insightfully pointed out (12 June 2004, comment 3), “very few researchers are looking at endogenous TRPC channels.” As one of those “few researchers” I would like to contribute my perspective to this E-conference:

The Drosophila phototransduction cascade is perhaps the oldest and best studied signalling pathway in which light stimulated, G-protein coupled, PLC activity ends with calcium influx through two well-defined classes of endogenous TRPC channels, TRP and TRPL. The total light-induced current in the fly eye is completely eliminated by protein null mutants in these two genes.

It has been known for over 15 years that phospholipaseC activity is essential for TRP and TRPL activation(analysis of norpA mutants); indeed it is fair to suggest that this finding was the conceptual basis for the widely accepted and largely undisputed idea that mammalian TRPC channels can be activated via phospholipaseC activity.

However, in the eye, despite a number of rigorous studies over the years, it has been difficult to understand the biochemical basis of this requirement for PLC activity (reviewed in detail elsewhere). In particular the requirement of InsP3 receptor (InsP3R) activity (with or without calcium release) in TRP and TRPL activation have been difficult to confirm.

Most recently and of immediate relevance to this debate we have analysed flies in which the only InsP3R in the completed version of the Drosophila genome has been deleted (complete gene missing). When analysed by patch-clamp electrophysiology the activation of TRP and TRPL is completely normal. This suggests that InsP3R activity is not required to activate TRPC channels in Drosophila photoreceptors.

In the meantime, although an enormous variety of thoughtful experiments have been done in mammalian cell lines to address the equivalent question, no one seems to have bothered to do the following experiment; analyse the activation of an endogenous TRPC channel in loss- of-function mutants in the three mammalian InsP3Rs within the context of a well-defined endogenous signalling cascade. Therefore with respect to the role of InsP3R activity in activating TRPC channels, until such an experiment is done, it is perhaps less meaningful to conclude that “Since TRPC channels have evolved quite significantly from fly, it is it is unreasonable to think that they would have to retain the same activation mechanisms that are used in fly.” (Randen Patterson, 12 June comment 4).

With regard to the potential role of lipid second messengers in activating TRPC channels I would once again like to place in perspective the analysis of this question in Drosophila phototransduction.

On the basis of the numerous studies that Roger Hardie and I have now published on this issue, it seems most reasonable to conclude that in the fly eye diacylglycerol kinase plays a crucial role in inactivating the light response (TRPC channel activity) during light induced PLC activity. What is less clear is how it does this,i.e the identity of the second messenger whose levels it regulates; candidates include diacylglycerol, its metabolites PUFA’s (polyunsaturated fatty acids), PIP4,5 or perhaps indeed more distant lipid metabolites that have so far not caught the attention of the calcium signalling community.

Until mammalian TRPC channel investigators can show that within the context of an endogenous signalling cascade (see paragraph above) diacylglycerol kinase does not play a role in inactivating TRPC channels it will be difficult to make “evolutionarly statements” about the equivalence or otherwise of the mechanisms by which lipid second messengers activate TRPC channels.

In summary it is worth reflecting that (1) Two key elements that underpin the mammalian TRPC signalling cascade namely the channels themselves and the requirement for phospholipaseC activity were discovered in the fly eye. (2) Until equivalent experiments have been done in mammalian systems, the evolution of different signalling mechanisms downstream of PLC activity in fly v mammals must remain an untested hypothesis.

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