In addition to the small family of three store-operated Orai channels, another somewhat larger family of ion channels are also linked to phospholipase C, although not exclusively to Ca2+
store depletion. These are a sub-family of the larger family of TRP channels, called TRPC for canonical TRPs, because they are most closely related in both structure and function to the founding Drosophila
photoreceptor channel, TRP (Montell et al., 2002
). In mammals, there are 7 TRPC channels, designated TRPC1 through TRPC7, although TRPC2 is a pseudogene in humans. As is the case for their Drosophila
counterparts, all appear to be activated downstream of phospholipase C. There may be two fundamental phospholipase C-mediated activation mechanisms for TRPC channels. The most widely accepted mechanism, and for which there is currently no debate, is a mechanism that depends upon the products of phospholipase C in some manner, but not upon the depletion of intracellular Ca2+
stores. This was most clearly demonstrated for Drosophila
TRP, which was efficiently activated by light (through phospholipase C) in flies lacking IP3
receptors (Acharya et al., 1997
). Trebak et al.
(Trebak et al., 2003a
) investigated the activation of mammalian TRPC3 channels ectopically expressed in kidney cell line (HEK293). In this system, TRPC3 channels were efficiently activated when phospholipase C was activated through muscarinic cholinergic receptors, but not when Ca2+
stores were depleted with thapsigargin. Injection of IP3
into the cells resulted in rapid and substantial release of Ca2+
stores, but no activation of TRPC3. Finally, blockade of IP3
receptors by intracellular application of heparin completely prevented the discharge of intracellular stores by a muscarinic receptor agonist, but did not prevent the activation TRPC3. Similar studies have provided similar evidence for other members of the TRPC family, with the possible exception of TRPC1, discussed in more detail below (Trebak et al., 2007
What then is the precise signal originating form phospholipase C activity that is responsible for TRPC channel activation? This question has not as yet been definitively answered. Since IP3
and vide infra store depletion seem not to be involved, the logical alternative is the other reaction product diacylglycerol. Indeed, Hofmann et al.
(Hofmann et al., 1999
) reported that TRPC6 channels could be directly activated by diacylglycerol, either by application of membrane permeable diacylglycerol analogs to intact cells, or by application of diacylglycerols to channels in excised patches. TRPC3, 6 and 7 all appear to behave similarly: they can all be activated independently of phospholipase C activity by diacylglycerols (Vazquez et al., 2004a
). This does not appear to involve protein kinase C, since activators of protein kinase C are potent inhibitors of TRPC channels (Trebak et al., 2005
;Venkatachalam et al., 2003
). Nonetheless, it is questionable as to whether the effect of diacylglycerol on the channels is direct. First, Smyth et al.
(Smyth et al., 2005
) showed that tyrosine kinase-coupled growth factors could increase the surface expression of TRPC3 channels; however, the newly expressed channels could not be activated by synthetic diacylglycerols, while those already present could. Second, the signaling tyrosine kinase, src, was shown to be essential for diacylglycerol activation of TRPC3, but was not required for the channel’s constitutive activity (Vazquez et al., 2004b
). Third, in contrast to the earlier report for TRPC6, Lemonnier et al.
(Lemonnier et al., 2008
) observed activation of single TRPC7 channels by synthetic diacylglycerols in cell attached mode, but this activation was lost upon excision of the patch.
TRP channels, including TRPC channels, appear to be comprised of a tetrameric assembly of TRPC molecules (Abramowitz & Birnbaumer, 2008
). Heteromeric assemblies of TRPC subunits can occur, at least experimentally, although perhaps with certain restrictions. Hofmann et al.
(Hofmann et al., 2002
) reported that, in keeping with their apparent common mode of activation, TRPC3, 6 and 7 readily combine to form heteromeric channels, but these TRPCs cannot combine with TRPC1, 4 or 5. Likewise, TRPC1, 4 and 5 can combine with one another, but not readily with TRPC3, 6 or 7. In one study combinations of TRPC1 with TRPC5 resulted in channels with distinct properties from homotetramers of TRPC5 (Strübing et al., 2001
). Note however, that other labs have sometimes reported combinations outside of these rules, based on co-mmunoprecipitation
There has been little work on TRPC2 because it is not a functional gene in humans; it may also be activated by diacylglycerol (Lucas et al., 2003
). TRPC1 is probably a special case, discussed below. TRPC4 and 5 are both clearly activated downstream of phospholipase C, but not by diacylglycerol.
The activation mechanism for TRPC4 and 5 has been difficult to resolve. It is rather clear that phospholipase C is important for their activation, as receptor activation of these TRPCs is blocked by a phospholipase C inhibitor. Schaefer et al.
(Schaefer et al., 2000
) demonstrated that TRPC4 and 5 (1) can be activated by either PLCβ or PLCγ-coupled receptors, but not by store depletion; and (2) activation was blocked by the PLC antagonist U73122. However, neither IP3
nor diacylglycerol activated these channels. Subsequently, other reports have provided evidence that Ca2+
(Blair et al., 2009
;Gross et al., 2009
) can strongly augment TRPC5 currents. Also, Gαi subunits have been reported to activate the channels (Jeon et al., 2008
). However, neither of these mechanisms can explain the general sensitivity of TRPC4 and 5 to PLC activation, whether through PLCβ or PLCγ. Also, the failure of the classical activator of SOC channels, thapsigargin, when GPCR activation works well, argues that Ca2+
cannot be the initiator of the activation mechanism.
One suggested mechanism for TRPC5 activation is the depletion of plasma membrane PIP2 (Trebak et al., 2009
). Generally, ion channels are activated or potentiated by polyphosphoinositides (Suh & Hille, 2005
;Hilgemann et al., 2001
) and this appears to be the case for TRP channels as well (Voets & Nilius, 2007
;Rohacs & Nilius, 2007
). This is also the case for the TRPC channels TRPC3, 6, 7 and 5 when studied in excised patch mode (Trebak et al., 2009
;Lemonnier et al., 2008
), despite the fact that in intact cells, these TRPC channels are turned on subsequent to PLC activation. One tool used in the study of PIP2 regulated channels is a small group of pharmacological inhibitors of the enzyme that phosphorylates PI to PIP, PI 4-kinase. The two most commonly used, wortmannin and LY294002, are potent inhibitors of the PI 3-kinase, but at a 10-fold higher concentration, they block PI 4-kinase (Linseman et al., 1999
;Linseman et al., 1998
). When applied in this concentration to cells expressing TRPC5, the compounds activated TRPC5-dependent [Ca2+
signals and TRPC5 currents (Trebak et al., 2009
). Under similar experimental conditions, TRPC3 channels were not activated. Yet, in excised patches, TRPC5 single channels were strongly activated by addition of PIP2 to the patch, and to a greater extent than that seen with receptor activation in intact cells. To explain these contradictory findings, Trebak et al.
(Trebak et al., 2009
) proposed two distinct functions of PIP2. PIP2 may associate directly with the channels and may be required for their activity, as appears to be the case for many other ion channels. In addition, PIP2 may have a signaling function which involves its interaction with a regulatory molecule. PIP2 binding to this regulator causes it to bind to the channels and inhibit their activity. Activation of PLC would specifically deplete the pool of PIP2 involved in braking TRPC5 activity leading to channel activation. In excised patches, loss of both pools of PIP2 results in a channel lacking the negative regulator, but also lacking the PIP2 having a necessary co-factor function for channel activity. Addition of PIP2 in this condition would be expected to produce strong channel activation, as was observe experimentally.
Interestingly, a similar difficulty exists for the founding member of the TRP superfamily, the photoreceptor TRP channel in Drosophila
). As for mammalian TRPCs, which are most closely related to the Drosophila
TRP is absolutely dependent upon PLC activity. Experimental evidence suggests that changes in the lipid composition of the plasma membrane, as a result of PLC activation, play at least a positive modulatory role in the activation of Drosophila
TRP. An intriguing hypothesis recently put forth by Huang et al.
(Huang et al., 2010
) is that TRP may be activated by the protons released in the hydrolysis reaction of PLC. As to whether this might play a role in the activation of mammalian TRPCs requires further investigation.