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E-Conference: Defining Calcium Entry Signals

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TRPC channels and the second messengers that regulate them

4 June 2004

Thomas Gudermann

More than 20 years have elapsed since the suggestion was made that receptor activation could lead to Ca2+ entry into smooth muscle cells by mechanisms independent of membrane depolarization, and the concept of receptor-operated Ca2+ entry was developed. Receptor-stimulated cation channels are gated in response to agonist binding to a cell membrane receptor that is molecularly distinct from the channel protein itself.

Over the past couple of years a large family of mammalian homologues of the Drosophila transient receptor potential (TRP) visual transduction channel have been identified. These channels, in particular those of the canonical TRPC subfamily, are likely molecular correlates of receptor-operated cation channels as defined before. In order to add some spice to the discussion, I shall refer to TRPC proteins primarily as receptor-operated cation channels, because in my personal view none of them has been rigorously proven to be a store-operated Ca2+ channel in a direct sense. Thus, although TRPCs like any Ca2+-permeable ion channel may have an impact on store-operated Ca2+ influx, there appears to be insufficient hard evidence to classify them as genuinely store-operated. Therefore, in my opinion a clear distinction can be made between receptor-operated and store-operated Ca2+ entry with respect to TRPC proteins as potential molecular correlates.

Receptor-operated cation entry plays an eminent physiological role in vascular smooth muscle cells. I shall, therefore, use this cell type as a kind of physiological springboard for further arguments and views. In vascular smooth muscle cells, cation influx through non-selective cation channels is thought to be required for cell membrane depolarization in response to vasoconstrictors resulting in the activation of voltage-gated Ca2+ channels, Ca2+ influx, and constriction of blood vessels.

As TRPC6 is highly expressed in vascular smooth muscle cells, it represents a likely molecular candidate for the vasoconstrictor-activated Ca2+-permeable cation channel. As characterized in native vascular smooth muscle cells, the latter channels are activated via phospholipase C (PLC)-coupled receptors and by diacylglycerol (DAG) independent of protein kinase C. Upon heterologous expression in CHO-K1 cells, TRPC6 behaves as a receptor-activated non-selective cation channel insensitive to the depletion of internal stores being activated by DAGs independent of protein kinase C.

To summarize, TRPC3, TRPC6, and TRPC7 form a structural and functional subfamily of DAG-sensitive cation channels coupling receptor/PLC signalling pathways to cation entry. Although these TRPC proteins are generally classified as the DAG-responsive subfamily, it is still a highly contentious issue as to whether DAG can be regarded as the physiological activator of native channel complexes, while there is no doubt that all members of the TRPC3/6/7 subfamily can principally be activated by DAG. As deduced from pharmacological inhibition of DAG lipase and DAG kinase, endogenously generated DAG is sufficient for channel activation. Most notably, receptor agonists and DAGs do not display additive effects on TRPC3 and TRPC6 current amplitudes, suggesting that the same TRPC channels are activated by DAG and by PLC-linked receptors and that DAG may be the decisive second messenger generated by PLC.

However, so far a direct interaction of DAG with TRPC3/6/7 proteins has not been demonstrated. Postulating such a direct contact between the lipid messenger and the channel protein as demonstrated for the interaction of capsaicin with TRPV1, possible interaction sites in the channel protein might be located within the first intracellular loop and neighbouring portions of transmembrane helices 2 and 3. In the absence of a mapped DAG-contact site in the channel protein, TRPC3/6/7 activation by C1 domain-containing proteins, such as chimaerins, MUNC13s, RasGRPs, and even DAG kinases, cannot be excluded and deserves experimental clarification.

Also, the role of PKC is more complex than initially assumed. Pharmacological inhibition of PKC, as well as enzyme down-regulation by long-term phorbol ester treatment, showed that PKC activity is not required for DAG-dependent TRPC3/6/7 activation. However, short-term PKC activation prior to DAG addition completely blocks channel gating, and PKC inhibition results in decreased TRPC deactivation. Thus, while not necessary for channel activation, PKC is intrinsically involved in TRPC channel regulation.

As yet, we cannot satisfactorily answer the question as to whether DAG alone is sufficient for TRPC3/6/7 activation. Phorbol ester and DAG treatment of many cells is frequently accompanied by the engagement of tyrosine kinase-dependent signaling pathways. In fact, the Src-family tyrosine kinase Fyn physically associates with TRPC6 and phosphorylates the protein thereby substantially enhancing channel activity. However, it has not been reported whether ablation of Fyn-dependent TRPC6 tyrosine phosphorylation negatively impacts on DAG-induced channel activation.

Finally, one may even pose the heretical question whether the subsummation of TRPC3/6/7 as the DAG-sensitive subfamily is correct at all. Recently, patch-clamp recordings on vomeronasal neuron dendrites provided evidence that TRPC2 may also be a DAG-gated cation channel. Along these lines, DAG-activated currents were observed in TRPC5-expressing HEK 293 cells. An important methodological difference between these recent findings and other studies on DAG-sensitive TRPCs is the use of electrophysiology, which is a highly sensitive technique, on the one hand, and fluorescence imaging on the other hand. Thus, it appears to be worth examining whether in fact all TRPCs respond to DAG, albeit with different potency and efficacy.

Another lipid messenger that has been implicated as a modulator of TRP channel function is PIP2. The latter phospholipid inhibits Drosophila TRP and TRPL, as well as mammalian TRPV1. On the contrary, constitutive TRPM7 activity is enhanced upon PIP2 addition and rapidly inactivated by PIP2 hydrolysis. Clearly, further experimentation is required to come up with a unifying hypothesis to reconcile the discrepant effects of PIP2 on various TRP channels.

Some TRP channels (TRPC6, TRPV2, C. elegans TRP-3) have been described to be rapidly translocated to the plasma membrane in response to various stimuli like G-protein-coupled receptor agonists (TRPC6), growth factors (TRPV2), and sperm activation (TRP-3). These observations give rise to the concept that some TRP ion channels may be held in reserve in intracellular vesicles. So far, however, a mechanistic understanding of the stimulus-induced translocation to the plasma membrane still remains elusive.

For the sake of brevity, I shall not go into detail to discuss the barrage of published data on the activation of TRPC proteins by store depletion. Suffice it to say, store-dependent and -independent gating mechanisms have been postulated for nearly each member of the TRPC family. However, there appears to be a kind of consensus allowing us to surmise that TRPC proteins are receptor-operated cation channels sharing a common gating mechanism that is contingent on PLC activation. Under particular circumstances -- defined by the endowment of a special cell line with signaling proteins, TRP protein expression level, species and methodology chosen -- certain TRPCs might be part of store-operated calcium entry channel complexes.

One important reason for the discrepant results is related to different methods used. Fluorescence imaging is quite an indirect measure of channel activity, because it mirrors the accumulated free Ca2+ concentration irrespective of the source. It may even reflect reversed- mode Na+/Ca2+ exchanger contribution rather than TRPC channel activity. As overexpression of TRPC proteins frequently results in constitutive Ca2+ entry, store depletion and recalcification protocols that do not account for this fact will inevitably lead to false positive results. Patch-clamp recording as a direct approach to monitor channel activity has the potential to overcome most of these difficulties, but is prone to deplete the cell of diffusible messengers that might regulate ion channel function. Ideally, several independent methodological approaches should be applied and the results be critically compared.

Because the composition of functional TRP channel complexes is largely unknown, it has proven difficult to ascribe receptor-activated cation currents to molecularly defined TRP proteins. Several studies have recently shown that various TRP channels can assemble as heteromeric complexes which differ in their biophysical properties. Systematic analyses on the principles of TRP channel formation offer the conceptual framework to assess the gating mechanism, regulation and physiological role of distinct TRP proteins in their native environment. So far, two principally different themes have evolved from TRP channel heteromultimerization: On the one hand, heteromultimerization can alter the biophysical properties of channel complexes whose individual members can also be functionally expressed alone (e. g. TRPV5/6), on the other hand, heteromultimerization is necessary to measurably transport certain TRP channels subunits to the cell membrane (e. g. TRPC1/5, TRPM6/7). In most instances, the daunting task to define the composition of channel complexes under physiological native conditions still remains to be addressed.

A last question that I think needs to be answered is: Why are there so many genetically and functionally related TRP proteins. For instance, why do we need three classical DAG-sensitive TRPC channels? Future experiments, preferably in vivo, will have to address the issue as to whether TRP proteins belonging to a certain subfamily are functionally redundant or not and whether unique and indispensable physiological roles can be ascribed to individual members. I am absolutely sure that answering these questions will keep the scientific community busy for quite some time.

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