1. Introduction to TRPC
The veil of transient receptor potential canonical (TRPC) channels was lifted by the search of mammalian homologs of the Drosophila TRP channel [1
], the photoreceptor required for visual transduction. As indicated by the term “canonical”, the TRPC family was first identified within the whole TRP superfamily because of their highest homology to the Drosophila TRP channel [7
This family is composed of seven members. Based on their sequences and properties, TRPC channels can further be divided into two subfamilies, with TRPC1/4/5 falling into one and TRPC3/6/7 into the other [8
]. Studies have indicated that TRPC2 is a pseudogene in humans [9
] and it will not be further discussed in this review. All TRPC channels share a common topology; they have six transmembrane domains (TM1-TM6) with a pore located between TM5 and TM6. Both the long N- and C-terminus are located intracellularly. N-terminus harbors several motifs like the ankyrin repeats, a coiled-coil domain, and a putative caveolin-binding region. C-terminus harbors the TRP signature motif, a calmodulin/inositol 1,4,5-trisphosphate (IP3
) receptor-binding (CIRB) region, and a PDZ-binding motif (unique to TRPC4/5). All these motifs are essential for the multimerization and trafficking of TRPCs, and the interaction of auxiliary proteins with TRPCs as reviewed elsewhere [7
A functional TRPC channel is formed by the association of four subunits. Depending on the composition, it can either be a homotetramer or a heterotetramer. TRPCs can heterogeneously multimerize not only with TRPCs but also with other TRP proteins. For instance, TRPC1-TRPP2 [10
], TRPC1-TRPV6 [11
], and TRPC1-TRPV4 [12
] heterotetramers have been reported. Through different combinations, heterotetramers are conferred distinct biophysical properties from that of homotetramers to further meet diverse physiological requirements [13
TRPCs are non-selective cation channels that are permeable to a multitude of monovalent and divalent cations, including Ca2+
]. Due to the importance of Ca2+
in different kinds of cellular activity, including gene expression, proliferation, differentiation, apoptosis, migration, secretion, and muscle contraction, TRPCs have received wide attention as reviewed elsewhere [15
]. The ubiquitous expression of these channels in different types of cell, tissue, and organ circumstantially confirm their biological importance [16
]. A substantial number of studies have been conducted to explore their functions under normal and pathological conditions and revealed their roles in cardiac hypertrophy [17
], vasoconstriction [18
], neointimal hyperplasia [19
], angiogenesis [20
], platelet activation [21
], myoblast differentiation [22
], salivary fluid secretion [23
], motor coordination [24
], podocyte dysfunction [25
], and respiratory rhythm regulation [26
]. All of these effects are mediated by the Ca2+
influx through these channels.
TRPCs may be the most elusive ion channels ever reported. In the early days when they were just discovered, some evidence implied that they are the potential store-operated calcium channels (SOCCs), but conflicting results and interpretations emerged later, making this identity controversial as reviewed elsewhere [27
]. On one hand, there were findings showing that STIM and Orai are essential players of the store-operated calcium entry (SOCE) [28
]. On the other hand, there were also studies showing TRPC channels are regulated by Orai [33
] or STIM [34
], thus conferring SOCC properties. The current view is that both Orai and TRPC would be activated by a decrease in ER [Ca2+
] that is detected by the ER-located STIM. Although Ca2+
signals generated by both Orai and TRPC overlap with each other, they regulate distinct functions in different cells. A recent review has thoroughly examined this [35
]. In fact, TRPCs are more commonly regarded as receptor-operated calcium channels (ROCCs). G protein-coupled receptors (GPCRs) on the plasma membrane sense the external stimuli such as hormones, neurotransmitters, and growth factors, leading to the activation of phospholipase C (PLC) through G protein; activated PLC catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2
) to IP3
and diacylglycerol (DAG). TRPC3/6/7 can be directly activated by DAG, while TRPC1/4/5 are activated through more complicated pathways, interacting protein partners and phosphorylation may both be involved in this process [14
]. It was found that scaffolding proteins such as the Na+
exchanger regulatory factor (NHERF) binds to TRPC4/5 and desensitize these two channels to DAG. TRPC4/5 only would become sensitive to DAG when NHERF is dissociated from TRPC4/5, which can be caused by depletion of PIP2
or inhibition of protein kinase C (PKC) [36
]. Apart from gating mechanisms like SOCCs and ROCCs, TRPC1, TRPC5, and TRPC6 were also reported to be mechanosensitive channels [39
]. On the other hand, some evidence suggested that this property is conferred or potentiated by GPCRs [42
Various signals input from GPCRs upstream of TRPCs and diverse biological effects triggered by Ca2+ influx downstream of TRPCs together make TRPCs the most versatile ion channels. These effects also increase the complexity of studies on TRPCs. To gain a better understanding of their behavior, plenty of studies have been carried out to explore the post-translational modifications (PTMs) and mutations of TRPCs. Both PTMs and mutations may modulate the properties of the channels under different conditions. Different kinds of PTM have been found in TRPCs; they include N-glycosylation, disulfide bond formation, ubiquitination, S-nitrosylation, S-glutathionylation, acetylation, and mostly, phosphorylation. These covalent modifications may change the activity, the subcellular location, or the protein-protein interaction of TRPCs, consequently modulating their function. Alterations of TRPC functions would further affect physiology and pathophysiology of different cells, tissues, and systems. Similarly important aspect is the natural mutations occurring in TRPCs. Natural mutations including deletion, frame shift, and substitution of TRPCs were reported; they result in gain-of-function or loss-of-function mutants that finally lead to inherited diseases. Here we try to compile all related reports of PTMs and mutations, aiming to provide an overview of the research in this field.
12. Concluding Remarks
TRPCs are versatile channels widely expressed in different types of cells and tissues; they sense various kinds of extracellular stimuli and relay them into different output responses. PTM is an important strategy used to modulate the channel’s properties, therefore allowing the cells to better meet different physiological requirements. An equally important aspect is the natural mutations found in TRPCs. These mutations may generate GOF/LOF mutants and cause maladaptive responses and/or inherited diseases. Figure 8
shows an overview of the signal transduction through TRPC channels.
TRPCs can be covalently modified by several types of PTM. In general, phosphorylation is the most common one, it may enhance or decrease the channel’s activity, affect the trafficking of the channel or the interaction of TRPCs with other proteins. According to the few reports that documented N-glycosylation of TRPCs, N-glycosylation tends to reduce the channel activity. A disulfide bond may help to stabilize the pore architecture and also to confer the sensitivity of the TRPCs to reducing reagents. TRPC5 can be subjected to S-nitrosylation or S-glutathionylation; this sensitivity to oxidation stress allows a brand-new regulation pathway for this channel. Ubiquitination causes internalization but not degradation of TRPC4. Acetylation is found in TRPC6, but its function is still unknown.
For natural mutations reported in TRPC channels, TRPC6 mutants and the related disease FSGS are the best studied. Most TRPC6 mutants are characterized to have GOF phenotype; they elevate the cytosolic Ca2+, and subsequently cause cell death and/or cytoskeleton disorganization of podocytes, resulting in FSGS. Another well-studied mutation is the TRPC3 mutant in moonwalker mice. The mutation also results in a GOF phenotype, impairs the growth and differentiation of Purkinje cells and leads to ataxia. SNPs of TRPC6 are found to be associated with diseases such as IHPS, IPAH, NPSLE and CFS. While most mutations or SNPs are linked to cellular dysfunction, it is intriguing that a missense SNP in TRPC4 has been found to decrease risk of MI in diabetic patients.
The traditional strategy to study PTM of proteins begins with prediction of modification site, followed by site-directed mutagenesis, and then expression and functional characterization. By this method, most PTMs of TRPCs have been identified and studied. Although this method provides the best functional annotation of each PTM, the drawback is that only a certain type of modification in a particular channel can be studied each time. On the other hand, by using the high-throughput, mass spectrometry-based method, a global and unbiased search of multiple types of PTM in a large population of proteins can be conducted. It has been proven that when using the high-throughput method, several novel PTM sites in TRPCs could be identified. The biggest drawback of high-throughput method is the lack of functional annotation of the identified PTM. High throughput methods and traditional methods are complementary to each other and can be used in conjunction to investigate PTMs of TRPCs comprehensively. In the future, it is ideal to screen for changes of PTMs under different physiological or pathological conditions by using the high-throughput method followed by a confirmation and characterization using traditional methods to better understand their influence on the TRPC channel proteins and on the cells.
Recently, the protein structure of TRPC3/4/5/6 have been resolved by cryo-EM with an atomic resolution [81
]. The detailed 3D structure offers a new way to evaluate potential residues that are critical for the gating and/or permeation of TRPCs. It would also be interesting and fruitful to revisit those missense or nonsense mutations or SNPs from public database, and to speculate and/or to investigate the possible effects of these mutations/SNPs on the TRPC proteins and on the cells.