Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel

147

105

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca 2؉ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca 2؉ store depletion. Apart from this mode of regulation, little is known about other receptordependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor-and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.In many types of cells, membrane receptors coupled to phospholipase C (PLC) 1 promote inositol 1,4,5-trisphosphate (IP 3 )-mediated release of Ca 2ϩ from endoplasmic reticulum and Ca 2ϩ entry across the plasma membrane through both capacitative or store-operated and non-capacitative calcium entry pathways (1-3). Although the molecular identity of capacitative and noncapacitative calcium entry channels, as well as the signaling mechanisms involved, remain uncertain, mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channel have been considered as potential candidates (4, 5), particularly members of the canonical TRP (TRPC) subfamily (designated TRPC1 through TRPC7 (6)). The human isoform of TRPC3, originally cloned by Zhu et al. (7), has been shown in many heterologous expression systems to behave as a receptoractivated channel that can be activated also by exogenous application of diacylglycerols (DAGs) independently of store depletion. In fact, DAG-induced activation of TRPC3 and its structural relatives TRPC6 and TRPC7 (8, 9) provides the likely mechanism of activation of these channels by phosphoinositide-specific PLClinked receptors, independently of IP 3 and store depletion. Apart from its regulation by DAG generated from PLC stimulation through either G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), little is known about additional signaling pathways downstream of receptor-stimulation involved in activation of TRPC3. Stimulation of either of these receptor pathways results in rapid tyrosine phosphorylation of cellular proteins (10, 11), and so in the present study we investigated whether tyrosine kinases might play a role in the signaling mechanism underlying receptor-and DAG-dependent activation of TRPC3. We found that inhibition of tyrosine kinase activity completely abrogated the ability of either GPCR st...

Section: Discussionmentioning

confidence: 44%

Section: Discussionmentioning

confidence: 44%

Obligatory Role of Src Kinase in the Signaling Mechanism for TRPC3 Cation Channels

Vázquez

Wedel

Kawasaki

et al. 2004

147

105

“…TRPC6 was first reported to be phosphorylated by PKA in human platelets, but this does not appear to alter 1-oleoyl-2-acetyl-glycerol-stimulated calcium influx (104). Similarly, PKA has been reported to inhibit TRPC5 but not TRPC6 currents when the proteins are expressed in 293T cells (105), and activation of TRPC6 by cAMP analogues is insensitive to H89 (99).…”

Section: Discussionmentioning

confidence: 99%

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

Chiluiza

Krishna

Schumacher

et al. 2013

Background: Signaling events affected by disease-associated mutations in TRPC6 are poorly defined. Results: Expression of mutant TRPC6 induces ERK1/2 activation via both cell-autonomous and non-cell-autonomous mechanisms. Conclusion: Mutant TRPC6 activates complex signaling pathways that lead to the release of paracrine factors activating ERK. Significance: Understanding the signaling pathways downstream of gain-of-function TRPC6 is crucial for understanding TRPC6-mediated biology and pathology.

“…Cyclic AMP-dependent protein kinase A potentiates TRPV1 by direct phosphorylation (27). TRPC6 is a substrate for protein kinase A, although phosphorylation appears to not affect cation permeation (28). The TRPM7 activity seems to be dependent on the Src family PTKs, although whether TRPM7 is directly phosphorylated by tyrosine kinases or not is unclear (29).…”

Section: Stimulation Of the Egf Receptor Induces Rapid Tyrosine Phospmentioning

confidence: 99%

Regulation of TRPC6 Channel Activity by Tyrosine Phosphorylation

Hisatsune

Kuroda

Nakamura

et al. 2004

181

136

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.Various growth factors or hormones can induce activation of phospholipase C (PLC), 1 production of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), and Ca 2ϩ influx across the plasma membrane (1, 2). This PLC-dependent Ca 2ϩ influx is thought to play important roles in many physiological functions, such as cell proliferation and apoptosis, T cell activation, and the maturation and functions of B cells (3). Therefore, it is important to understand regulation of such PLC-dependent Ca 2ϩ channels, because modulation of the channel activities can profoundly affect these various physiological processes. The transient receptor potential (TRP) channel superfamily has emerged as candidates responsible for such a PLC-dependent Ca 2ϩ influx. The TRP channel superfamily can be divided into at least three subfamilies of Ca 2ϩ -permeable nonselective cation channels (TRPC, TRPV, and TRPM families), having closely related structures comprised of six transmembrane domains, a large NH 2 -terminal cytoplasmic domain, and a COOH-terminal cytoplasmic domain (4). Among the three subfamilies, TRPC channels are one of the molecules that have been extensively characterized.The TRPC channel family is composed of seven non-selective ion channels that can be divided into four subgroups (TRPC1; TRPC4 and -5; TRPC3, -6, and -7; and TRPC2) based on their amino acid sequences and functional similarities (4 -6). Recent investigations have extensively studied the regulation of TRPC channel activity. TRPC1, -4, and -5...