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date: 23 January 2020

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Abstract and Keywords

The Drosophila Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. These channels are evolutionarily conserved from yeast to mammals and are found in many organisms and tissues. The TRP family is classified into seven subfamilies, while the most closely related to the Drosophila TRP are members of the TRPC (Canonical) subfamily. This review focuses on a comparison between properties of Drosophila TRP, discovered in the native photoreceptor cells, and that of mammalian TRPC channels. These properties include: (i) organization of TRP channels in multimolecular signaling complexes via PDZ-containing scaffold proteins, (ii) mutations causing constitutive activity of TRP channels and cell degeneration, (iii) regulation of TRP channels by phosphorylation, and (iv) hypoxia/anoxia-activation of TRP channels. Hence, we suggest that knowledge gained from studies of Drosophila may guide studies in mammals that attempt elucidating diverse types of diseases caused by TRPC channel malfunction.

Keywords: TRPC channels, Drosophila TRP and TRPL, INAD and NHERF scaffold proteins, TRP phosphorylation, constitutive activity, hypoxia sensing


It is now widely recognized that TRP channels were discovered in the study of Drosophila eye. Cosens and Manning reported on a spontaneously occurring mutant, which they called “Type A,” with stimulus-intensity–dependent behavioral and electrophysiological anomalies (Cosens & Manning, 1969). With a dim light stimulus, the electroretinogram (ERG: extracellularly recorded summed responses of the eye) looked nearly normal, but under a continuous bright light stimulus, the response decayed to, or nearly to, the baseline. This finding was potentially interesting to us because the mutant phenotype might suggest a defect in phototransduction (the process by which light signals are converted to electrical signals), and we were starting to isolate potential phototransduction mutants (Pak, 2010; Pak, Grossfield, & White, 1969). However, the report did not generate widespread interest at the time.

For one thing, the cellular origin of the ERG components was not well established at the time: it was not clear at what signaling level the mutation caused the defect. It could affect the activation of photoreceptors, or be involved in the chain of events subsequent to photoreceptor activation. In fact, Cosens and Manning themselves alluded to the possibility of “a breakdown in a transmitter system,” though it was not clear what this transmitter system might mean in the present-day context. They also suggested that the defect might be in the visual pigment turnover. In addition, the report was based on the study of a single allele. The phenotypes of different alleles in a given gene can be very different, and studies based on a single allele need to be interpreted with caution.

The most direct way to determine whether the abnormal ERG response originated in the photoreceptor was to record directly from the photoreceptors. The only way of doing this at the time was by intracellular recording. Fortunately, we had developed the techniques for recording intracellularly from Drosophila photoreceptors (Alawi & Pak, 1971; Wu & Pak, 1975). Intracellular recordings showed unequivocally that the phenotypes seen in the ERG were present in intracellular recordings as well, suggesting that the defect in the mutant originated in the photoreceptors (Minke, 1982; Minke, Wu, & Pak, 1975; Figure 1A).

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 1The phenotype of the trp mutant is mimicked by lanthanum (La3+) in wild-type fly.

A. Intracellular recordings from single photoreceptor cell of white-eyed trpCM raised at 24oC showing voltage responses to increasing intensities of orange lights (in relative log scale).

B. Intracellular recordings from a single photoreceptor cell of white-eyed Musca domestica in response to increasing intensities of orange lights, as indicated. The left column shows control responses; the middle column shows the responses of the same cell 1 min after La3+ injection to the extracellular space; and the right column shows partial recovery of the response 20 min after La3+ injection.

From Minke, 1982; and Suss Toby et al., 1991.

These authors also measured the amount of photopigment activated by light in the mutant fly during the decline of the response to the low steady-state level. They found that, during the decay phase, only a small fraction of pigment was activated, ruling out the possibility that the mechanism for the mutant phenotype was due to a defect in photopigment turnover (Minke, 1982; Minke et al., 1975).

As for the single-allele issue, this objection could be countered by isolating additional alleles and showing that results from these alleles are consistent with those obtained with the allele discovered by Cosens and Manning. As we mentioned, a few years before the study by Cosens and Manning appeared, we began to isolate mutants defective in the ERG response using chemically mutagenizing, isogenized, Oregon R wild-type stock for studying Drosophila phototransduction (Pak, 2010; Pak et al., 1969). We mutagenized each major chromosome separately, the third chromosome (where trp is localized) being the last to be mutagenized. Nevertheless, by 1974, we began recovering mutants with similar ERG phenotypes, which did not complement the mutant discovered by Cosens/Manning. Ultimately, we isolated nine allelic mutants, including a true null mutant, trpP343 (Scott, Sun, Beckingham, & Zuker, 1997) and a constitutively active mutant, trpP365 (Yoon et al., 2000). These mutants were freely made available to other investigators. The results showed that, indeed, the phenotypes of allelic mutants of this gene do vary, but the phenotype described by Cosens and Manning represented the general features of many mutant alleles of this gene. With these results on hand, and in consultation with Dr. Cosens, we decided to name the Type A mutant transient receptor potential (trp, [Minke et al., 1975]) and this particular allele, trpCM.

The trp gene was cloned and molecularly characterized by Montell and Rubin (1989) and shortly afterward by Wong and colleagues (1989), for a detailed summary of trp cloning and sequencing (see Minke, 2010). This was an important achievement, as it allowed the cloning of trp-related genes, ultimately leading to the identification of a new superfamily of trp genes. However, cloning of this gene did not lead immediately to the recognition of its function as an ion channel, or to the enzymatic cascade leading to its activation. Sequencing revealed that the trp gene encodes a 1275 amino-acid membrane protein with no homologies to any known protein in the database. It had eight transmembrane domains (later revised to six transmembrane [TM] and a pore domain) and displayed many topological features reminiscent of receptor-transporter-channel proteins. However, the possibility that it might encode a light-activated channel was ruled out because light responses were present in mutants that showed no protein product in Western blot analyses (Montell & Rubin, 1989; Wong et al., 1989).

The nature of TRP function began to emerge in the following several years. Hochstrate showed that application of the non-specific Ca2+ channel blocker, lanthanum (La3+), to the extracellular space of the blowfly Calliphora retina caused a dramatic decline in the receptor potential during light, making a wild-type receptor potential of Calliphora resemble that of a trp mutant (Hochstrate, 1989). These results suggested to Minke and colleagues a potential clue to TRP function (Suss Toby, Selinger, & Minke, 1991). They verified the preceding observations in three species of flies and proposed that the stated effect of La3+ might arise from its ability to block a Ca2+ transporter protein, which normally allows Ca2+ entry into photoreceptors to cause photoreceptor excitation and to replenish the internal Ca2+ pool (Suss Toby et al., 1991; Figure 1B).

In subsequent review papers, Minke and Selinger, (Minke & Selinger, 1991, 1992) further elaborated on their “conformational coupling model,” which is based on that of Berridge (Berridge, 1995). In this model, the TRP protein was seen as a Ca2+ transporter/channel, which oscillates between conducting and non-conducting states through interactions with IP3 receptor protein, which also acts as a Ca2+ sensor of the filling state of the IP3-sensitive Ca2+ stores. By this time, it had already been established that phototransduction in Drosophila utilizes a phosphoinositide-mediated cascade (Bloomquist et al., 1988; Devary et al., 1987; Selinger & Minke, 1988), in which Gq-activated (Blumenfeld, Erusalimsky, Heichal, Selinger, & Minke, 1985; Devary et al., 1987) phospholipase C (PLC) plays a central role. This was established in large part through biochemical analyses of light-activated Drosophila and Musca signaling proteins and the mutants isolated in the screen mentioned previously. Therefore, it seemed reasonable to assume that both IP3 and IP3-receptor proteins could also be involved. Subsequently, it was shown that the IP3 receptor has no role in phototransduction in Drosophila (Acharya, Jalink, Hardy, Hartenstein, & Zuker, 1997; Raghu, Colley, et al., 2000; but see Kohn et al., 2015). Nevertheless, the model correctly highlighted the major role of TRP channel in light-mediated Ca2+ entry.

Application of the patch clamp technique to Drosophila photoreceptors (Hardie, 1991; Ranganathan, Harris, Stevens, & Zuker, 1991; for the method, see Katz, Gutorov, Rhodes-Mordov, Hardie, & Minke, 2017) led to the evidence that the trp gene encodes the major component of the light-activated channels (Hardie & Minke, 1992). Whole-cell patch clamp recordings showed that the light-activated channels of Drosophila are Ca2+ permeable (Hardie, 1991), and this finding was later extended by using Ca2+ selective microelectrodes and microfluorimetry by Ca2+ indicator fluorescence in whole-cell recordings (Peretz, Sandler, Kirschfeld, Hardie, & Minke, 1994; Peretz, Suss-Toby, et al., 1994). Ion permeability measurements led to the finding that the primary defect in trp mutants or in La3+ treated wild-type photoreceptors is a drastic reduction in the Ca2+ permeability of the light-sensitive channels themselves (Hardie & Minke, 1992). These authors concluded that the light response of a wild-type photoreceptor consists of two distinct conductances: one is highly Ca2+ permeable and is encoded by the trp gene, and the other, encoded by another gene, is responsible for the residual current in trp mutants. The nature of the second conductance was clarified by the work of Kelly and colleagues (Phillips, Bull, & Kelly, 1992). While searching for calmodulin-binding Drosophila proteins, they discovered a membrane protein, which they called “TRP-like” (TRPL) with homology to TRP (~40% overall identity and 74% identity within the transmembrane segments). They showed that it is smaller than TRP (900 vs. 1275 amino acids), has two calmodulin-binding domains (vs. one in TRP) in the C-terminal region, and an ankyrin repeat domain in the N-terminal region. Their analysis further revealed a transmembrane topology reminiscent of voltage-gated channels: six transmembrane segments, S1–S6, and a putative pore region between S5 and S6, except that the charged residues in S4 of voltage-gated channels are replace by non-polar ones. Moreover, within S5 and S6, they found several short stretches of amino acid identical to the mammalian voltage-gated Ca2+ channels. They suggested that both trp and trpl genes encode light-activated channels (Phillips et al., 1992).

The results of Hardie and Minke and those of Kelly and colleagues dovetailed each other in showing that TRP and TRPL channels together contribute to the light-activated conductance of Drosophila photoreceptors. A subsequent important study by Zuker and colleagues (Niemeyer, Suzuki, Scott, Jalink, & Zuker, 1996) isolated a null trpl allele, trpl302, and showed that the light response is largely abolished in the double mutant, trpl302;trpP301. The residual response in the double mutant arose from trpP301, which is not a complete null allele. It disappeared in the presence of low concentrations of La3+, which would block TRP but not TRPL channels. Subsequently, Zuker and colleagues (Scott et al., 1997) identified a true null trp mutant, trpP343, among the trp mutants Pak and colleagues had previously isolated. They showed that the light response is completely abolished in the double mutant, trpl302;trpP343. This series of experiments convincingly demonstrated that light-activated conductance consists solely of TRP and TRPL channels, and no other channels appear to contribute.

Thus, Drosophila TRP became the founding member of the TRP ion channel superfamily. The first mammalian members of the family were cloned by homology to Drosophila TRP (Wes et al., 1995; Zhu, Chu, Peyton, & Birnbaumer, 1995). We now know that TRP channels are widely evolutionarily conserved from yeast to mammals, are found in almost all organisms and tissues. They were classified into seven subfamilies: TRPC (classical or canonical), TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin), TRPN (NOMPC), TRPML (Mucolipin), and TRPP (Polycistin). The most closely related to Drosophila TRP and TRPL are members of the TRPC subfamily, typified by the ones cloned by Montell and colleagues (Wes et al., 1995) and by Birnbaumer and colleagues (Zhu et al., 1995) (the TRPC1).

In retrospect, it is now possible to evaluate the contribution of the original trpCM allele to the TRP field. In spite of the difficulties to the TRP field stemming from using the trpCM allele, it was a crucial tool for cloning and sequencing of the trp gene. The mapping of the trp locus to the edge of the third chromosome (Levy, Ganguly, Ganguly, & Manning, 1982; Wong, Hokanson, & Chang, 1985) was conducted on trpCM. The subsequent rescue of the trpCM phenotype (i.e., the transient receptor potential phenotype) by P-element-mediated germline transformation (Montell, Jones, Hafen, & Rubin, 1985) led to cloning and sequencing of the Drosophila trp gene (Montell & Rubin, 1989; Wong et al., 1989). In spite of this important contribution to the TRP field, several specific properties of the trpCM severely hampered recognizing the TRP protein as a light-activated ion channel, which probably prevented a widespread interest in TRP at that time. Specifically, electrophysiological analysis of the trpCM mutant revealed that its single-photon responses (quantum bumps) were normal (Minke et al., 1975). However, Western blot analysis suggested that the TRP protein is absent in trpCM (Montell & Rubin, 1989).

The main reason for this confusion arose from the fact that trpCM turned out to be a developmental temperature-sensitive allele (Minke, 1983) that expresses ~30% functional TRP at 19oC (Figure 2B) (Yoon et al., 2000), while at the restrictive temperature of 24oC, it was almost null. This property led to light-induced generation of normal quantum bumps when trpCM was raised at 19oC, the temperature used for bump analyses, and to the generation of a pronounced light-induced current (LIC; Reuss, Mojet, Chyb, & Hardie, 1997) (Figure 2A, bottom) and yet the receptor potential declined towards baseline during intense prolonged light.

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 2Functional analysis of the developmental temperature sensitive trpCM mutant by whole-cell recordings and Western blot analysis.

A. Intense light stimulation elicited no responses in the trpl302;trpCM double null mutant, when raised at 25°C (upper trace), while a large LIC was elicited from the same double mutant when raised at 19°C (bottom trace), indicating that trpCM is a developmental temperature sensitive trp mutant, showing a null phenotype when raised at 25°C, but not when raised at 19°C. Note that the light induced current (bottom trace) is truncated.

From Reuss et al., 1997.

B. Western blot analyses showing the expression level of the TRP channel in wild-type (lane 1) and trpCM/trpCM homozygot raised at 19°C (lanes 5) or 25°C (lane 6) flies. Note, the highly reduced expression of trpCM/trpCM in both conditions compared to WT flies and the reduction in expression level between 19°C and 25°C.The other lanes appear again in Figure 4B

This review does not intend to give a comprehensive outline of functional properties of TRPC channels. Recommended recent excellent reviews on TRPC channels are the reviews of Svobodova and Groschner (2016); Voolstra and Huber (2014); Dietrich, Kalwa, and Gudermann (2010); Takahashi, Kozai, and Mori (2012); and Birnbaumer (2009). We will concentrate on some features of the TRPC channel subfamily, which have been investigated in detail in Drosophila photoreceptors. The advantage of Drosophila photoreceptors is that the physiological role of TRP/TRPL channels as the light-activated channels has been unequivocally established. Therefore, the effects of deleterious mutations or post-translational modifications on these channels can guide and provide insight into similar studies on mammalian TRPC channels.

In Vivo Mutation Analyses Provide Insights into Properties of TRPC Channels

Pak and colleagues had carried out a mutagenic screen, using the alkylating agent ethyl methane sulfonate (EMS) as mutagen, for visually defective mutants by searching for defects in phototaxis or ERG response (Pak et al., 1969; Pak, 2010). The ERG screening included the use of a specific light stimulating protocol that induced the Prolonged Depolarizing Afterpotential (PDA) (Pak, Shino, & Leung, 2012; Minke, 2012). For this reason all flies had their screening pigments removed genetically to allow the development of PDA and avoid any potential complications associated with the pigments. In the PDA protocol, the flies were illuminated with intense blue light to induce large photopigment conversion (Minke, 2012; Pak, Shino, & Leung, 2012). Such stimulation protocol brings the phototransduction cascade to its upper limit of activation for an extended time, during which millions of molecules of signaling proteins are activated (Minke, 2012). Therefore, any reduction in a molecularly-dependent functional process required for the generation of maximal PDA, such as biogenesis of rhodopsin or biogenesis and proper insertion of the TRP channel into the membrane of the signaling compartment (the rhabdomere) would lead to an abnormal PDA. Hence, the PDA screen has a great power for isolating visual mutants with a clear phenotype.

The Discovery of Multi-Molecular Signaling Complexes

The genetic visual screen and particularly the PDA screen, have produced a large number of visual mutants, which helped researchers discover key proteins that participate in invertebrate phototransduction with wide implications for general signaling mechanisms. One such discovery was the isolation of the inaD mutant, InaDP215, which was isolated using the PDA screen (Pak, 1995) and was subsequently cloned and sequenced by Shieh and Niemeyer (Shieh & Niemeyer, 1995).

The Drosophila PDZ Scaffold Protein, Inactivation but no Afterpotential D (INAD)

The inaD gene encodes a 674 amino acid protein that was found to be highly enriched in the photoreceptor cells. The original sequence analysis revealed two protein interacting motifs called “PDZ (PSD95, DLG, ZO1) domains” (Shieh & Niemeyer, 1995). These domains are recognized as protein modules that bind to a variety of signaling, cell adhesion, and cytoskeletal proteins by specific binding to target sequences, typically, though not always, in the final three residues of the C’-terminus (Shieh, Zhu, Lee, Kelly, & Bahiraei, 1997). Immunoprecipitation technique showed that INAD binds two proteins, one of which was the TRP channel. InaDP215 harboring a single missense mutation, M422K, which disrupt the TRP–INAD interaction (Shieh & Zhu, 1996). The INAD-interacting domain on the TRP channel was localized at first to the last 19 residues of its C’-terminus but later was suggested to compose only the last three residues (Chevesich, Kreuz, & Montell, 1997; Shieh et al., 1997). Studies on Calliphora have extended these findings, showing that INAD binds not only TRP but also the No Receptor Potential A (NORPA), which encodes Phospholipase Cβ (PLCβ) and the Inactivation but no Afterpotential C (INAC), which encodes an eye specific Protein Kinase C (eyePKC) (Huber, Sander, & Paulsen, 1996). The interaction of INAD with TRP, PLCβ, and eyePKC was later confirmed in Drosophila, and a thorough analysis of the protein sequence revealed that it contained five PDZ domains instead of two (Tsunoda et al., 1997; Figure 3). Subsequent studies suggested that, in addition to PLCβ, eyePKC, and TRP, other signaling molecules such as calmodulin (CaM), the major rhodopsin (RH1), TRPL, and Neither Inactivation Nor Afterpotential C (NINAC), which encodes for a myosin III, also bind to the INAD signaling complex (Chevesich et al., 1997; Xu, Choudhury, Li, & Montell, 1998). However, such diverse binding partners without physiologically demonstrated functions must be dynamic.

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 3Schematic representation of the INAD protein complex.

The INAD sequence contained five consensus PDZ domains (indicated by numbers 1–5) and identified specific interactions between PKC (INAC, encoded by the inaC gene) and PDZ2 (or PDZ4), TRP and PDZ3, and PLC (NORPA, encoded by the norpA gene) with PDZ1 and PDZ5. This binding pattern is still in debate due to several contradictory reports. It was also reported that the INAD contains a Ca2+-calmodulin binding site, which may be involve in its regulation. It also binds the actin cytoskeleton via myosin III (NINAC, encoded by the ninaC gene).

From Katz & Minke, 2009.

The identification of INAD as a scaffold protein provides a mechanism for the co-localization of major phototransduction components in spatial proximity. However, the functional role of the INAD protein was unknown at that time. Using immunofluorescent staining, the function of INAD (Tsunoda, Sun, Suzuki, & Zuker, 2001) and TRP (Chevesich et al., 1997; Xu et al., 1998) in the localization of the major phototransduction proteins was examined. Accordingly, the localization of the signaling proteins INAD, TRP, NORPA, INAC, NINAC, and RH1 was studied using the InaD or trp null mutant flies. The results showed that the INAD is correctly localized to the rhabdomeres in the inaC null mutants (where eyePKC is missing) and in norpA virtually null mutant (where PLCβ is virtually missing), but severely mislocalized in the null trp mutant, thus indicating that TRP, but not NORPA or INAC, is essential for localization of the signaling complex to the rhabdomere. Furthermore, in the absence of INAD, the TRP, NORPA, INAC, but not the NINAC or RH1, were mislocalized to the cell body, showing that the INAD protein is essential for the retention of the bound PLCβ and INAC signaling proteins to the rhabdomere (Tsunoda et al., 2001; Xu et al., 1998). The study of these mutants was also used to show that TRP and INAD do not depend on each other to be targeted to the rhabdomeres. Thus, INAD–TRP interaction is not required for targeting but for anchoring and retention of the signaling complex (Tsunoda et al., 2001). Additional experiments on TRP, NORPA, and INAD further showed that INAD has an important function in preventing NORPA degradation (Xu et al., 1998), which is especially important for response termination due to the GTPase activating protein (GAP) activity of NORPA (Cook et al., 2000).

A structural study of INAD has suggested that the binding of signaling proteins to INAD may be a dynamic process that constitutes an additional level of phototransduction regulation (Mishra et al., 2007). This study showed two crystal structural states of isolated INAD PDZ5 domain, differing mainly by the presence of a disulfide bond. This conformational change has light-dependent dynamics that were demonstrated by the use of transgenic Drosophila flies expressing an INAD having a point mutation that disrupts the formation of the disulfide bond. In this study, a model was proposed in which eyePKC phosphorylation at a still-unknown site promotes the light-dependent conformational change of PDZ5, distorting its ligand-binding groove to PLCβ and thus regulating phototransduction. Further studies showed that the redox potential of PDZ5 is allosterically regulated by its interaction with PDZ4 (Liu et al., 2011). Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ4-5 “supramodule” locks PDZ5 in the reduced state by raising the redox potential of a disulfide bond. Acidification, potentially mediated via light-dependent PLCβ hydrolysis of Phosphatidylinositol 4,5 bis phosphate (PIP2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP channel (Liu et al., 2011). However, demonstration of the physiological significance of these light-dependent changes in INAD is still lacking.

The Mammalian PDZ Scaffold Protein NHERF Interacts with TRPC4 and TRPC5 Channel Proteins

Studies have shown that some TRPC channels are organized in supra-molecular complexes similar to the INAD signaling complex. The scaffolding protein Na+/H+-exchanger regulatory factor 1 (NHERF) was isolated as a co-factor required for inhibition of type 3 Na1/H1 exchanger by protein kinase A, and was localized to the renal brush-border (Yun et al., 1997). Later the NHERF protein was found to interact with TRPC4/5. The NHERF protein family is composed of NHERF1 (also known as ERM-Binding Protein 50 [EBP50]) and NHERF2, which shares 44% sequence homology. These proteins contain two PDZ domains and a sequence at the C’-terminus that binds several members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adapters (Ardura & Friedman, 2011; Dunn & Ferguson, 2015; Murthy et al., 1998; Terawaki, Maesaki, & Hakoshima, 2006). In a biochemical study, it was shown that the first PDZ domain of NHERF binds murine TRPC4 or TRPC5 as well as PLC-β1 and PLC-β2 (Tang et al., 2000). The interaction of PLC-β1, TRPC4, and NHERF was demonstrated in the HEK293 cell line stably expressing TRPC4, and in adult mouse brain by co-immunoprecipitation experiments. Since NHERF binds also to the cytoskeleton via ERM proteins, the cytoskeleton seems to be part of this supramolecular organization (Tang et al., 2000). The binding of two partners to the same PDZ domain suggests that NHERF can form a homodimer via PDZ2 and the PDZ1 domains, bringing TRPC4 or TRPC5 in vicinity of the PLCβ1 and PLCβ2 (Suh, Hwang, Ryu, Donowitz, & Kim, 2001; Tang et al., 2000; for a review, see Constantin, 2016).

The last three C-terminal amino acids (TRL) of TRPC4 compose a PDZ-interacting domain that binds to the scaffold protein EBP50. In order to explore the role of TRPC4–EBP50 interaction on the subcellular localization of TRPC4, a truncated TRPC4 lacking the last three amino acids was examined. Accordingly, immunofluorescence microscopy analysis showed that wild-type TRPC4 channels were distributed evenly on the cell surface, while the TRPC4 mutant lacking the PDZ motif accumulated into cell outgrowths with a punctate distribution pattern. Cell surface biotinylation revealed a 2.4-fold reduction in plasma membrane expression of the truncated TRPC4 mutant compared to wild-type TRPC4. Furthermore, the consequences of the interaction between NHERF and the membrane-cytoskeletal adaptors of the ERM family on cell surface expression of TRPC4 was examined. In cells co-expressing TRPC4 and a NHERF mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated NHERF in a perinuclear compartment and in vesicles associated with actin filaments. Hence, the TRPC4-NHERF-ERM complex regulates TRPC4 localization and surface expression in transfected HEK293 cells (Mery, Strauss, Dufour, Krause, & Hoth, 2002). The effects of EBP50–TRPC5 interaction on the activity and cellular distribution of TRPC5 were also investigated. In this study, rat TRPC5 (rTRPC5) and a mutant TRPC5 with a deletion of the PDZ-binding domain Val, Thr, Thr, Arg, Leu (VTTRL)were examined in the HEK293 cell expression system (Obukhov & Nowycky, 2004). Accordingly, both wild-type and mutant TRPC5 were localized to the plasma membrane, and deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel, when studied with patch-clamp technique. Co-expression of EBP50 with rTRPC5 led to a significant delay in the time-to-peak of the histamine-evoked, transient, large inward current. However, EBP50 did not modify the activation kinetics of the VTTRL-deletion mutant.

Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes and co-localized in some cells of the renal medullary descending vasa recta (DVR). Co-immunoprecipitation experiments from renal medullary lysates indicated physical interaction of TRPC4 and NHERF-2 proteins (Lee-Kwon, Wade, Zhang, Pallone, & Weinman, 2005). These results suggest that the scaffold protein NHERF-2 assembles with TRPC4 in renal medullary DVR.

Together, the interaction of EBP50 (NHERF) with TRPC4 or TRPC5 channels has different effects on the cellular distribution and activation of the channels in different tissues.

Interaction of the Mammalian PDZ-Scaffold Protein NHERF, with TRPC4 and TRPC5 Is Required for DAG Activation

In a recent study, it was demonstrated that TRPC4 and TRPC5 sensitivity to diacylglycerol (DAG) is dependent on the association of NHERF to the TRPC at the C’-terminus. Accordingly, TRPC4/5 becomes DAG sensitive when NHERF is dissociated from these TRPC channels. The interaction of NHERF and TRPC4/5 is dependent on PKC phosphorylation at the C-terminal. Strikingly, DAG sensitivity was achieved under several experimental paradigms, such as: (i) PKC inhibition, (ii) removal of a C-terminal PKC phosphorylation site in the PDZ-binding motif, (iii) NHERF1 and NHERF 2 down-regulation, (iv) co-expression of a NHERF1 mutant (NHERF1–E68A) incapable of interacting with the C-terminal of TRPC5, and (v) co-expression of Gq/11-coupled receptors. Importantly, C-terminal conformational rearrangements engendered by PIP2 depletion were also required. The experiments of this study based on electrophysiology, co-immunoprecipitations, and intermolecular Fluorescence Resonance Energy Transfer (FRET) have thus suggested that a crucial step in TRPC5 activation is the dissociation of NHERF proteins from the channel C’-terminus, conferring DAG sensitivity (Storch et al., 2017). Collectively, C-terminal NHERF and PIP2 interaction stabilize a DAG-insensitive channel conformation. During receptor activation, PIP2 level at the plasma membrane is reduced by PLC, resulting in an active TRPC5 conformation, characterized by C-terminal rearrangements and the ensuing dissociation of NHERF1 and NHERF2, thereby conferring DAG sensitivity on TRPC4 and TRPC5 channels. The possibility that a similar mechanism may lead to DAG sensitivity of Drosophila TRP has not been investigated (see the section “Anoxia Activation of Drosophila TRP/TRPL Channels in the Dark” in this chapter).

The Mammalian Caveolin-1 Scaffold Protein Interacts with TRPC1, TRPC4, and TRPC3

Caveolae are glycosphingolipid- and cholesterol-enriched membrane microdomains found in many vertebrate cells, which are enriched with Caveolin, a transmembrane scaffolding protein. Caveolin-1 interacts with TRPC1, TRPC4, and TRPC3 channels via binding to both the N’ and C’ termini. A caveolin-1 conserved binding motif was identified in all TRPC members at the N-terminal part close to the first transmembrane domain TM1 (amino acids 271–349 or 322–349). Deletion of this region prevented the targeting of TRPC1 to the plasma membrane and exerted a dominant negative effect on endogenous inward current induced by intracellular Ca2+ store depletion, designated Store Operated Calcium Entry (SOCE). The expression of truncated caveolin-1 (Cav1Δ51-169), lacking its protein scaffolding and membrane-anchoring domains, disrupted plasma membrane targeting of TRPC1 and suppressed thapsigargin- and carbachol-stimulated Ca2+ entry (Brazer, Singh, Liu, Swaim, & Ambudkar, 2003).

Using the caveolin-1 (Cav1)-deficient mice (Razani & Lisanti, 2001), it was shown that in endothelial cells that the scaffolding protein governs the localization and interactions of TRPC1 and TRPC4. Furthermore, Cav-1 is associated with a dynamic protein complex consisting of TRPC4, TRPC1, and IP3 receptors (IP3Rs), while the loss of Cav-1 impairs the localization of TRPC4 and ACh-mediated calcium entry (Murata et al., 2007). In general, caveolae are thought to organize a multiprotein calcium signaling complex containing TRPC1, which is anchored to caveolin-1 and is associated with signaling proteins such as the IP3R, calmodulin (CaM), plasma membrane calcium pump (PMCA), and Gαq/11 (Ambudkar & Ong, 2007; Lockwich et al., 2000).

Although Caveolin-1 appears to play a role in the interaction of mammalian TRPC with a variety of signaling proteins, and to underlie their retention in the plasma membrane regions, similar functions of Caveolin have not been investigated in Drosophila, and therefore are not elaborated on in this review.

Mutations Causing Constitutive Activity of TRPC Channels

Many TRP channels exhibit constitutive activity, which is mostly observed in cell-based expression systems. This constitutive activity can lead, in many cases, to cellular degeneration, which can be readily observed morphologically and by biochemical assays. In cell-based expression system, it is difficult to know if the constitutive activity is physiologically relevant. In the Drosophila photoreceptor cells, the TRP channels are closed in the dark and open upon illumination. Therefore, the isolation of the Drosophila trpP365 mutant showing constitutive activity of the channel in the dark has raised a widespread interest.

The Drosophila trpP365 Mutant Fly Shows Constitutive Activity of the TRP Channel

The P365 mutant isolated in the EMS screen of Pak and colleagues was highly unusual because it showed an extremely fast retinal degeneration phenotype even at the pupa stage, and unreliability of the complementation test due to its semi-dominant nature. Later, the P365 mutation was mapped to the edge of chromosome 3R, an area that also harbors the trp locus. Electron micrograph (EM) studies of P365 retinae showed light-independent retinal degeneration, while heterozygote flies showed normal morphology (Figure 4A), but illumination induced retinal degeneration. Initially, the P365 mutant was not identified as a trp allele because trp alleles did not show fast retinal degeneration. Also, most trp mutants did not show significant TRP channel protein expression in Western blot analysis, unlike the P365 mutant at both homozygote and heteroallelic combination (Figure 4B). In addition, ERG measurements showed that, although the sensitivity to light of the P365 mutant was highly reduced in both homozygote and heterozygote mutant flies, still it did not show the typical transient receptor potential (trp) phenotype (Yoon et al., 2000). In order to show that the P365 mutant phenotype is caused by mutations in the TRP channel, a transgenic fly carrying the mutation of P365 only in the trp locus (P[TrpP365]) was constructed. The P[TrpP365] on Wild Type (WT) background showed reduced light sensitivity, similar to that found in the P365 mutant fly, supporting the notion that mutations in the TRP channel caused the observed phenotype. Whole-cell current measurements during voltage steps from photoreceptor cells revealed similarity between currents of P365 mutant measured in darkness (Figure 4D) and light-induced wild-type TRP currents. Since 10 µM La3+ blocked these currents (Figure 4E), it further supported the notion that the TRP channels of the P365 mutant are constitutively opened in the dark (Figure 4C–D). Sequencing the TrpP365 gene revealed four missense mutations: P500T, H531N, F550I, and S867F (Yoon et al., 2000). It was therefore important to determine which of the mutations cause the P365 phenotype. Using transgenic flies harboring different combinations of these mutations, it was possible to determine the F550I mutation as causing the P365 phenotypes (Hong et al., 2002). In conclusion, the TrpP365 mutant fly harbors a missense F550I mutation (Figure 4F) in the TRP channel, which causes constitutive activity, retinal degeneration and highly reduced sensitivity to light. It was unclear, however, whether the retinal degeneration is a consequence of massive Ca2+ influx through the constitutively active TRP channels, or if the mutation causes retinal degeneration, which promotes the constitutive activity of the channels. Using over-expression of the sodium–calcium exchanger, CalX, it was shown that the retinal degeneration of TrpP365/+ could be partially rescued (Liu et al., 2007). Hence, elevated extrusion of Ca2+ rescues the degeneration, supporting the notion that retinal degeneration is a consequence of massive Ca2+ influx through the constitutively active TRP channels.

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 4Functional analysis of the TrpP365 mutant by whole-cell recordings electron microscopy and Western blot analysis.

A. Electron micrographs (EM) of transverse sections through the ommatidial layer (at the level of R7 photoreceptor nuclei) of TrpP365/trpCM (left) and TrpP365/TrpP365 (right), both raised at 19°C. All samples were obtained from newly enclosed adult flies, and all flies were on a w (white-eyed) background. The TrpP365/TrpP365 mutant retina appears highly degenerated, and the degeneration was delayed in the heteroallele TrpP365/trpCM. Scale bar, 1 µm.

B The P365 mutant shows large channel protein expression in both homozygote and heteroallelic combination. Western blot analyses showing the expression level of the TRP channel in heteroallelic mutant TrpP365/trpCM raised at 19°C (lanes 3) or 25°C (lane 4) and controls: wild-type (lane 1), TrpP365/TrpP365 homozygotes (lane 2), TrpP365/+ heterozygotes (lane 7). All raised at 25°C

C. A typical light-induced current (LIC) of a wild-type cell (left trace) in response to an orange stimulus, and the absence of any responses in TrpP365/trpCM and TrpP365/TrpP365 (middle and right traces, respectively). The duration of the orange light stimulus is indicated above each trace.

D. Families of current traces elicited in the dark from photoreceptor cells by a series of voltage steps of 10 mV from -100 mV to +80 of wild-type (left column), the light-insensitive cells TrpP365/trpCM (middle column), and TrpP365/TrpP365 homozygotes (right column). Membrane currents were recorded 30 s after establishing the whole-cell configuration with physiological concentrations (1.5 mM) of Ca2+ in the bath. The initial holding potential was -20 mV. Note the outward current in the dark in the TrpP365/trpCM and TrpP365/TrpP365 mutants (but not in wild-type fly) due to the constitutive activity of the mutant channel.

E. Application of 10 µM La3+ to the bath suppressed the dark voltage elicited membrane currents.

Traces B-F are from Yoon et al., 2000.

F. Multiple sequence alignment of TRP (NP_476768.1), TRPL (NP_476895.1), TRPC1 (NP_001238774.1), TRPC3 (NP_001124170.1), TRPC4 (NP_003297.1), TRPC5 (NP_036603.1), TRPC6 (NP_004612.2), and TRPC7 (NP_065122.1) as measured by GeneiousTM alignment using the Blosum62 cost matrix. Shown: the amino acid multiple sequence alignment at the S4–S5 loop and part of S5 G540, D545, K548—yellow, and F550—cyan, of TRP and the corresponding amino acids in TRPL and in their human counterparts are highlighted. The numbering represents the position of the last amino acid.

The TRP F550I mutation is located at S5 (transmembrane helix number 5) adjacent to the S4–S5 loop, and it is conserved in TRPL (F557, Figure 4F). This amino acid position shows only hydrophobic conservation among mammalian TRPC channels, while the two flanking Phe residues are more conserved (see sequence alignment, Figure 4F). Interestingly, two independent studies using a random chemically induced mutation in Drosophila TRP (Hong et al., 2002; Yoon et al., 2000) and a high-throughput mutagenic screen of TRPV1 (Myers, Bohlen, & Julius, 2008) both found mutant channels where this position was mutated and gave rise to constitutively active channels. In both cases, the mutation induced cell death, and in the case of Drosophila, the mutation resulted in retinal degeneration (Figure 4A). The exact mechanism of how those mutations cause constitutive channel activity is still unknown. Nevertheless, the TrpP365 mutation turned out to be extremely useful for introducing Ca2+ into Drosophila photoreceptors in the dark in a variety of in vivo studies. In these studies, elevated cellular Ca2+ triggers important cellular processes such as light- (and Ca2+)-dependent TRP channel dephosphorylation (Voolstra et al., 2017), and light- (and Ca2+)-dependent TRPL translocation (Meyer, Joel-Almagor, Frechter, Minke, & Huber, 2006; and see further in this chapter).

N-Linked Glycosylation of TRPC3 and TRPC5 Causing Constitutive Activity

N-linked glycosylation, the enzymatic reaction in which proteins are converted into glycoproteins, occurs at Asn residues within a specific sequence context (Asn-X-Ser/Thr, where X represents any amino acid except proline [Pless & Lennarz, 1977]). The effect of different N-glycosylation patterns on the function of TRPC channels was compared between TRPC3 and TRPC6. These channels, together with TRPC7, constitute a TRPC subfamily, whose members are activated by DAG in a membrane-delimited fashion (Hofmann et al., 1999). TRPC6 reveals a very low constitutive basal activity. In contrast, TRPC3 reveals pronounced constitutive basal activity in the absence of a receptor agonist. TRPC6 reveals glycosylation sites in both heterologous expression systems and in pulmonary vascular smooth muscle cells (PASMC), where TRPC6 is involved in PASMC proliferation (Weissmann et al., 2006). Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln), deleting one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of wild-type and mutant TRPC6 channels expressed in HEK293 cell revealed that TRPC6 is dually glycosylated within the first (designated e1) and second (designated e2) extracellular loops as opposed to the monoglycosylated TRPC3 channel (Vannier, Zhu, Brown, & Birnbaumer, 1998). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics similar to that of TRPC3. Reciprocally, construction of an additional second glycosylated site in TRPC3, to mimic the glycosylation pattern of TRPC6, markedly reduced TRPC3 basal activity. Usually N-glycosylation affects protein folding, intracellular trafficking, or membrane targeting. For the case of TRPC3 and TRPC6 channels, the glycosylation mutant channels were inserted into the plasma membrane, indicating that the effect is on channel activity. Thus, the glycosylation pattern plays a pivotal role for the tight activation of TRPC6 through phospholipase C-activating receptors (Dietrich et al., 2010).

Functional Roles of TRPC Phosphorylation

The activity of many proteins is regulated by phosphorylation and dephosphorylation reactions. Protein kinases and phosphatases that are activated during neuronal activity orchestrate cellular events that ultimately reshape the neuronal events via phosphorylation and dephosphorylation of various ion channels, including many members of the TRP channel superfamily (Por, Gomez, Akopian, & Jeske, 2013; Voolstra, Bartels, Oberegelsbacher, Pfannstiel, & Huber, 2013; Voolstra, Beck, Oberegelsbacher, Pfannstiel, & Huber, 2010) (for a comprehensive review see (Voolstra & Huber, 2014). However, the physiological roles of phosphorylation and dephosphorylation in controlling TRP channel activity are largely unclear (Cao, Cordero-Morales, Liu, Qin, & Julius, 2013).

The Roles of Drosophila TRP and TRPL Phosphorylation

The Drosophila TRP channel revealed a considerable number of phosphorylation sites. Some of them directly affect the functional properties of the channel. The eye-specific protein kinase C (eyePKC, INAC), which is part of the INAD complex (see preceding discussion) was shown to phosphorylate the Drosophila TRP in vitro (Huber et al., 1996; M. Liu, Parker, Wadzinski, & Shieh, 2000). In contrast, the TRPL channel revealed lower number of phosphorylation sites, which to date were not found to affect directly the functional properties of the channel.

TRP Phosphorylation

Using quantitative mass spectrometry, 28 TRP differential phosphorylation sites from light- and dark-adapted flies were identified by Huber, Voolstra, and colleagues (Voolstra et al., 2013; Voolstra et al., 2010).

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 5Light-dependent phosphorylation of Drosophila TRP and TRPL channels.

(A) A model of Drosophila TRP and its phosphorylation sites. Amino acid residues that undergo phosphorylation are shown as circles. Sites that undergo enhanced phosphorylation in the light are shown as white circles; sites that undergo enhanced phosphorylation in the dark are shown as black circles; sites that revealed no significant difference in phosphorylation between light- or dark-adapted flies or that could not be assessed quantitatively are shown as gray circles (ex = extracellular; in = intracellular).

(B) A model of Drosophila TRPL and its phosphorylation sites.

From Voolstra & Huber, 2014.

Twenty-seven phosphorylation sites resided in the C-terminus, while a single site resided at the N-terminus. Fifteen of the C-terminal phosphorylation sites exhibited enhanced phosphorylation in the light, whereas a single site, Ser936, exhibited enhanced phosphorylation in the dark (Voolstra et al., 2013; Figure 5A). To investigate light-dependent TRP phosphorylation, phospho-specific antibodies were generated to specifically detect TRP phosphorylation at Thr849; Thr864, which become phosphorylated in the light; and at Ser936, which becomes dephosphorylated in the light (see following discussion). To identify the stage of the phototransduction cascade that is necessary to trigger dephosphorylation of Ser936 or phosphorylation of Thr849 and Thr864, phototransduction-defective Drosophila mutants and the phospho-specific antibodies were used. Strong phosphorylation of Ser936 in dark-adapted WT flies was observed, and weak phosphorylation in light-adapted WT flies was detected. Conversely, weak phosphorylation of Thr849 and Thr864 was observed in dark-adapted WT flies, and strong phosphorylation was observed in light-adapted wild-type flies. Additionally, in phototransduction-defective mutants, with highly reduced light response, strong phosphorylation of Ser936 and weak phosphorylation of Thr849 and Thr864 were observed, regardless of the light conditions. Conversely, a mutant expressing a constitutively active TRP channel (trpP365, see preceding; Hong et al., 2002) exhibited weak phosphorylation of Ser936 and strong phosphorylation of Thr849 and Thr864, regardless of illumination. These data indicate that, in vivo, TRP dephosphorylation at Ser936 and phosphorylation at Thr849 and Thr864 depend on the phototransduction cascade, but activation of the TRP channel and most likely Ca2+ elevation are sufficient to trigger this process (Voolstra et al., 2013).

To identify kinases and phosphatases of Thr849, Thr864, and Ser936, a candidate screen using available mutants of kinases and phosphatases that are expressed in Drosophila eye was applied. It was found that Thr849 phosphorylation was compromised in light-adapted inaC null mutants. Diminished phosphorylation in light-adapted PKC53e mutants was also found; suggesting that these two PKCs synergistically phosphorylate TRP at Thr849. Using a similar method, the S936 site was found to undergo light-dependent dephosphorylation by the rhodopsin phosphatase Retinal Degeneration C (RDGC). Accordingly, light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels but maintained a light–dark phosphorylation dynamic. These findings suggest that RDGC is one, but not the only, phosphatase involved in S936-TRP dephosphorylation (Voolstra et al., 2017).

Electroretinogram (ERG) measurements of the frequency response to oscillating lights in vivo was performed (Voolstra et al., 2017). Dark-reared flies expressing wild-type TRP (trpWT) exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation (Figure 6A-B). It was further found that preventing phosphorylation of the S936-TRP site by Ala substitution in transgenic Drosophila (trpS936A) abolished the difference in frequency response between dark- and light-adapted flies, resulting in high-frequency response also in dark-adapted flies (Figure 6C-D). In contrast, inserting a phospho-mimetic mutation by substituting the S936-TRP site to Asp (trpS936D) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies (Voolstra et al., 2017; Figure 6E-F). In addition, measurement of the response latency, using whole-cell voltage clamp, showed that trpS936A had a shorter latency compared with trpWT and trpS936D transgenic flies (Katz et al. 2017). Together, these studies indicate that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions.

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 6Frequency response amplitude to oscillating light in dark- and light-adapted wild-type and transgenic flies in which TRP phosphorylation is prevented at specific sites. Representative short segments of electroretinogram (ERG) responses to intense (near saturation) oscillating 527 nm light of 70 Hz, measured 6 s after light onset in dark-adapted (DA) and light-adapted (LA) fly strains: (A) Wild type (WT), (C) trpS936A, and (E) trpS936D transgenic flies in which the S936 site is replaced with Ala (preventing dephosphorylation) or Asp (mimicking constant phosphorylation), as indicated. B, D, F) The amplitude of the Fourier transform of the ERG responses to oscillating light of 70 Hz, measured 6 s after light onset in dark-adapted (DA) and light-adapted (LA) flies, is presented. The Fourier transform was calculated from time segments of 200 ms, 6 s after oscillating light onset. The graphs plot the Fourier transform of ERG responses as a function of frequency obtained from light- and dark-adapted fly strains, as indicated. A prominent peak at 70 Hz is observed in all light-adapted fly strains except for the trpS936D transgenic fly. However, in dark-adapted flies, only the trpS936A fly showed a pronounced peak at 70 Hz.

From Voolstra et al., 2017.

Phosphorylation of TRPL

Using mass spectrometry, nine phosphorylated Ser and Thr residues were identified in TRPL channels (Cerny et al., 2013). Eight of these phosphorylation sites resided within the C-terminus, and a single site, Ser20, was located at the N-terminus (Figure 5B). Relative quantification revealed that Ser20 and Thr989 exhibited enhanced phosphorylation in the light, whereas Ser927, Ser1000, Ser1114, Thr1115, and Ser1116 exhibited enhanced phosphorylation in the dark. The phosphorylation state of Ser730 and Ser931 was light-independent. To further investigate the function of the eight C-terminal phosphorylation sites, the Ser and Thr residues were mutated either to Ala, eliminating phosphorylation (TRPL8x), or to Asp, mimicking phosphorylation (TRPL8xD). The mutated TRPL channels were transgenically expressed in R1-6 photoreceptor cells of flies as trpl-eGFP fusion constructs. The mutated channels formed multimeres with WT TRPL and produced electrophysiological responses when expressed in trpl;trp double null mutant background indistinguishable from responses produced by WT TRPL. These findings indicated that TRPL channels devoid of their C-terminal phosphorylation sites form fully functional channels and argue against a role of TRPL phosphorylation in channel-gating or regulation of its biophysical properties. Since TRPL undergoes light-dependent translocation (Bähner et al., 2002), subcellular localization of the phosphorylation-deficient as well as the phospho-mimetic trpl-eGFP fusion constructs were analyzed. Dark-adapted TRPLWT-eGFP show marked eGFP signal in the rhabdomere, while in light-adapted conditions the eGFP signal translocate to the cell body. In contrast, the TRPL8x-eGFP displayed markedly different translocation from WT TRPL-eGFP. After initial dark adaptation, a faint eGFP signal was observed in the cell body, but no eGFP signal was present in the rhabdomeres. After 16 hours of light adaptation, a strong eGFP signal was observed in the cell body as in WT flies. This indicated that TRPL8x-eGFP fusion construct was newly synthesized during light adaptation. After four hours of dark adaptation, TRPL8x-eGFP was present in the rhabdomere, but 20 hours later, only faint eGFP fluorescence was observable in the cell bodies, and none in the rhabdomeres (Cerny et al., 2013).

In conclusion, the localization of light and dark phosphorylation sites in both TRP and TRPL channels is well established. However, the physiological roles of these post-translation modifications need further studies.

Regulation of Mammalian TRPC Channels by PKC Phosphorylation

Phosphorylation plays an important role in regulation of mammalian TRPC channels. The activation of TRPC3, TRPC6, and TRPC7 by the DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), is reversed by the PKC activator 12-myristate 13-acetate (PMA (Okada et al., 1999; Trebak, St J Bird, McKay, Birnbaumer, & Putney, 2003; Venkatachalam, Zheng, & Gill, 2003; Zhang & Saffen, 2001)).

PKC Phosphorylation of TRPC3, TRPC6, TRPC7, and TRPC5

Application of the PKC activator Phorbol 12-myristate 13-acetate (PMA) was shown to increase the phosphorylation of TRPC3 in vivo (Trebak et al., 2003), while mutating Ser712 to Ala in TRPC3, identified by comparison of conserved putative PKC phosphorylation sites between TRPC3, TRPC6, and TRPC7 channels, abolished both the PMA increase in phosphorylation and PKC-mediated inhibition of the channel (Trebak et al., 2005). Hence, the phosphorylation of Ser712 by PKC negatively regulates the activity of TRPC3.

The negative regulation of TRPC3 by PKC phosphorylation was also suggested to explain the phenotype of the moonwalker mouse mutant. The moonwalker mouse harbors a missense, gain-of-function mutation Thr635Ala in TRPC3, which causes cerebellar ataxia and abnormal development of cerebellar Purkinje neurons (Becker et al., 2009). In the cerebellum, TRPC3 channels expressed in Purkinje cell underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation (Hartmann et al., 2008; Hartmann et al., 2014). In these cells, TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long-term depression (LTD) underlying cerebellar motor learning (Hartmann et al., 2008; Hartmann et al., 2014). Using in vitro kinase assay, it was shown that Thr635 is phosphorylated by PKCγ. This result raised the hypothesis that the gain-of-function phenotype observed in the TRPC3-Thr635A mutant was a consequence of a decrease in PKC-mediated inhibition of the mutated channel lacking the phosphorylation site (Becker et al., 2009). However, neither the phosphorylation event at Thr635 nor the inhibitory physiological consequence have been confirmed in vivo. Furthermore, native TRPC3-dependent currents elicited in cerebellar Purkinje cells were not inhibited by conventional PKC or PKG kinases, arguing against the hypothesis that the gain-of-function phenotype is due to lack in phosphorylation-dependent inhibition of the TRPC3 channels (Nelson & Glitsch, 2012). A recent study using computational modeling and functional characterization supports a mechanism by which hydrogen bonding of Thr635 plays a significant role in maintaining a stable, closed state of the channels (Hanson, Sansom, & Becker, 2015). However, we are still lacking the hydrogen bonding partner of Thr635 for further support of this hypothesis in the mechanism underlying the gain of function of the moonwalking mouse and its relationship to Thr635 Ala substitution.

Activation of the mouse TRPC5 by carbachol via the muscarinic receptors (Zhu et al., 2005) results in rapid desensitization of TRPC5, which is blocked by inhibitors of PKC. Mutation of the putative PKC phosphorylation site Thr972Ala of TRPC5 resulted in a large decrease of carbachol-mediated desensitization of the channel. TRPC6 is also inhibited by PKC-mediated phosphorylation (Zhang & Saffen, 2001), while phosphorylation by Fyn (the Src family protein kinase) increased its activity (Hisatsune et al., 2004). TRPC6 phosphorylation by Ca2+-calmodulin-dependent kinase II (CaMKII) is required for channel activation, as inhibition of CaMKII prevented channel activation by carbachol (Shi et al., 2004).

Together, phosphorylation of TRPC channels by a variety of protein kinases is important for regulating channel activity. However, this regulation affects a variety of functional channel properties, which are different for Drosophila and mammalian TRPC channels.

Hypoxia/Anoxia-Sensing TRPC Channels

It was initially found in Drosophila eye that several processes that induced metabolic stress, rapidly activated the TRP and TRPL channels in the dark in vivo. The robust effect of metabolic stress revealed a rare property of the TRP channel proteins. Although it is unlikely that depletion of ATP is the physiological mechanism underlying TRP and TRPL activation, this striking phenomenon provides an insight into the physiological properties of TRP and TRPL channels and also, into specific mammalian TRPC channel.

Anoxia Activation of Drosophila TRP/TRPL Channels in the Dark

The activation of TRP and TRPL channels in the dark in vivo can be induced by applying continuous N2 flow on the living intact fly, causing anoxia. Accordingly, ERG measurements from intact flies showed opening of the channels in the dark by anoxia, which was completely reversible upon termination of the anoxic condition (Agam et al., 2000; Figure 7). The openings of the light-sensitive channels in the dark could also be obtained in photoreceptor cells of isolated ommatidia by application of mitochondrial uncouplers or by omitting ATP from the recording pipette. Furthermore, the effects of illumination and all forms of metabolic exhaustion were additive. Effects similar to those found in wild-type flies were also found in strong hypomorph mutant alleles of rhodopsin, Gq-protein, or phospholipase C, while genetic elimination of both TRP and TRPL channels prevented the effects of anoxia, mitochondrial uncouplers, and ATP depletion, thus demonstrating that the TRP and TRPL channels are sensitive targets of metabolic stress (Agam et al., 2000). In the presence of Ca2+, ATP depletion or inhibition of protein kinase C activated the TRP channels in the dark, while photo-release of caged ATP or application of the PKC activator PMA antagonized channel openings as measured by whole cell recordings. Furthermore, Mg2+-dependent stable phosphorylation events by ATPγS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels, either by light or by metabolic inhibition. The attenuation of the light response and channel openings by metabolic inhibition was also observed when Ca2+ was highly buffered by 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). However, subsequent application of Ca2+ to the extracellular medium (in the presence of 7.5–10 mM BAPTA in the recording pipette) combined with ATP depletion induced a robust and fast-rising dark current that was reminiscent of light responses (Agam, Frechter, & Minke, 2004).

TRPC Channels—Insight from the Drosophila Light Sensitive Channels

Figure 7Anoxia activated the TRP and TRPL channels in both WT and the PLC null mutant (norpAP24), as monitored by ERG and Ca2+-selective microelectrodes.

Extracellular voltage change (ERG, top traces in black) and potentiometric measurements with Ca2+-selective microelectrode (ECa, bottom traces in red) in response to orange lights (blue pulses (LM) and anoxia N2 green), in WT (upper) Drosophila and norpAP24 (lower) mutant, are shown. Note that there is no response to light in the norpAP24 mutant, and the initial slow phase of the electrical response to anoxia due to accumulation of K+ is missing in the Ca2+ signals of both the WT and the mutant. The calibrations for the ERG records are indicated in black, and the calibrations for the potentiometric measurements with the Ca2+-selective microelectrode are indicated in red.

From Agam et al., 2000.

A specific mechanism for activation of the TRP and TRPL channels by metabolic inhibition in the dark has been proposed (Hardie, Martin, Chyb, & Raghu, 2003; Raghu, Usher, et al., 2000). According to this mechanism, metabolic inhibition primarily attenuates DAG kinase activity due to ATP depletion, leading to a failure of DAG inactivation via prevention of DAG phosphorylation, resulting in channel activation by DAG accumulation. A support for the hypothesis that DAG activates the TRP and TRPL channels comes from the analysis of the rdgA mutant fly lacking DAG kinase (Masai, Okazaki, Hosoya, & Hotta, 1993), in which the TRP and TRPL channels remain constitutively open in the dark and do not respond to light (Hardie et al., 2003; Raghu, Usher, et al., 2000). Following experiments on the rdgA mutant, it was suggested that DAG, or its metabolites, polyunsaturated fatty acids (PUFAs), are second messengers of excitation (Hardie, 2003). In support of this model, the LIC of mutants with minimal PLC activity and very small light response are partially rescued when combined in a double mutant with rdgA (Raghu, Usher, et al., 2000). Furthermore, PUFAs, the lipid product of DAG lipases, activate the native TRP/TRPL channels in Drosophila photoreceptors and TRPL in heterologously expressed systems (Chyb, Raghu, & Hardie, 1999). However, so far, it has proven to be virtually impossible to activate the TRP and TRPL channels in normally light-responding photoreceptor cells by exogenous application of DAG or its surrogates (but see Delgado, Muñoz, Peña-Cortés, Giavalisco, & Bacigalupo, 2014).

In summary, activation of the TRP and TRPL channels by metabolic inhibition in the dark has been a useful tool to decouple the activity of the TRP and TRPL channels from the phototransduction machinery and to study their properties in isolation in the native system. Furthermore, these studies show that a combined action of cellular Ca2+ and experimental conditions that presumably inhibit DAG kinase, causing DAG accumulation lead to channel activation suggesting that DAG is required for channel activation. It is not clear, however, if activation of the channels by metabolic inhibition is equivalent to the mechanism underlying physiological activation of the channels by light.

Hypoxia-Sensing TRPC6 Channels in Pulmonary Smooth Muscle Cells

Regional alveolar hypoxia causes local vasoconstriction in the lungs, shifting blood flow from hypoxic to normoxic areas, thereby matching blood perfusion to alveolar ventilation and optimizing gas exchange. This mechanism is known as “hypoxic pulmonary vasoconstriction” (HPV; Jeffery & Wanstall, 2001; Wanstall, Jeffery, Gambino, Lovren, & Triggle, 2001). In isolated pulmonary arteries and isolated perfused lungs, the HPV response is typically biphasic. The first phase is characterized by a fast-transient vasoconstrictor response that starts within seconds and reaches a maximum within minutes. The second phase is characterized by a sustained pulmonary vasoconstriction. Severe hypoxia of 1% O2 was shown to induce cation influx and currents in pulmonary arterial smooth muscle cells (PASMC) from wild-type mice, which were absent in Trpc6 knockout mice. The acute HPV response in isolated pulmonary arteries was missing in Trpc6 knockout, but not the second sustained vasoconstrictor response, nor the pulmonary vasoconstriction response elicited by thromboxane mimetic using U46619. These experiments strongly suggest that TRPC6 is involved in acute HPV response in isolated pulmonary arteries. However, recombinant TRPC6 expressed heterologously cannot be activated by hypoxia (Weissmann et al., 2006), suggesting that hypoxia does not activate the TRPC6 channels directly. This same situation was also observed for the Drosophila TRPL channel expressed heterologously. Accordingly, anoxic conditions and mitochondria uncouplers activated the TRPL channels in intact Drosophila eye and isolated photoreceptors, respectively, while mitochondria uncouplers did not activate TRPL channel expressed heterologously ( unpublished data).

Hypoxia-induced TRPC6 activation in smooth muscle cells is probably mediated by PLC activation and thereby DAG accumulation. This suggestion was supported by a subsequent study in which the DAG analog OAG increased normoxic vascular tone in lungs from wild-type mice in isolated perfused and ventilated mouse lungs, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG, as well as the DAG kinase inhibitor R59949, reduced the strength of acute HPV in a dose-dependent manner, whereas thromboxane mimetic-induced vasoconstriction using U46619 was not reduced. Similar to OAG, R59949 mimicked HPV, since it induced vasoconstriction during normoxic ventilation in a dose-dependent manner. In contrast, the PLC inhibitor U73122, which consequently should block DAG production, inhibited acute HPV, whereas U73343, the inactive analog of U73122, had no effect on HPV. These findings support the hypothesis that DAG induces TRPC6-dependent acute HPV. The use of TRPC6-KO mice has thus provided important insights into the role of TRPC6 in normal physiology and disease states of the pulmonary vasculature. Manipulation of TRPC6 function may offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange (Fuchs et al., 2011; for a recent review, see Malczyk et al., 2017).


The founding member of the TRP family of channels, the Drosophila light-activated TRP channel, shares a large structural and functional similarity with the mammalian TRPC subfamily. Both the Drosophila TRP channel and some members of the mammalian TRPC subfamily are assembled into a multi-molecular signaling complex. The Drosophila INAD scaffold protein assembles the TRP, PLC, and eyePKC, while the mammalian NHERF assembles the TRPC4/5, PLC, and cytoskeleton. The mammalian Cav-1 protein is also associated dynamically in a protein complex consisting of TRPC4, TRPC1, and IP3 receptors. The scaffold proteins in both Drosophila and mammalian TRPC channels co-localize the signaling proteins adjacent to the channels. In addition, they are involved in the retention and targeting of the protein complex to the signaling surface membrane: in Drosophila, the binding to INAD prevents degradation of TRP, PLC, and eyePKC and therefore maintains a one-to-one stoichiometry among the signaling proteins, which is crucial for normal function (Cook et al., 2000). In the case of mammalian TRPC channels, the role of Cav-1 and NHERF is also to target TRPC channels to the plasma membrane, while NHERF also regulates the sensitivity to DAG of TRPC4/5 (Storch et al., 2017).

There is a striking similarity in the activation of Drosophila TRP/TRPL (Agam et al., 2000) and mammalian TRPC6 (Fuchs et al., 2010; Malczyk et al., 2013) by anoxia/hypoxia. Likewise, a similar underlying mechanism has been proposed for both, by which reduction of cellular ATP levels by hypoxia attenuates DAG kinase activity, thereby resulting in DAG accumulation and channel activation (Raghu, Usher, et al., 2000; Fuchs et al., 2010; Malczyk et al., 2013). The available eye-specific DAG kinase mutant, rdgA, in Drosophila showing activation of the TRP and TRPL channels in the dark, was very useful for reaching this conclusion. The critical role of DAG in mammalian TRPC channel activation has been clearly established (Hofmann et al., 1999; Storch et al., 2017). In contrast, in Drosophila, except for extremely slow activation of TRP channels by DAG in isolated rhabdomeric preparations used for single-channel recordings (Delgado et al., 2014), application of DAG to isolated ommatidia has failed to activate these channels due to a still-unclear reason. Nevertheless, several experimental observations suggest that DAG may play a similar excitatory role in both mammalian and Drosophila TRPC channels. These observations include:

  1. i. Constitutive activity of TRP channels (most likely) due to DAG accumulation in the rdgA mutant (Raghu, Usher, et al., 2000).

  2. ii. Presumed accumulation of DAG following application of metabolic inhibitors/anoxia (Agam et al., 2000), and conversely, suppressed TRP channel activity upon ATP application (Agam et al., 2004; Delgado et al., 2014).

  3. iii. The enhanced excitation by application of DAG lipase inhibitor in tissue culture cells expressing TRPL (Lev, Katz, Tzarfaty, & Minke, 2012).

Most studies on TRPC channels have been conducted in recent years on mammalian TRPC channels. The main reason probably stems from the diverse and important physiological functions of mammalian TRPC channels (Bandyopadhyay et al., 2005; Dietrich et al., 2005; Neuner et al., 2015; Poteser et al., 2006; Poteser et al., 2011; Quick et al., 2012). Moreover, a relatively large number of physiological disorders and diseases arises from malfunction of these channels (Fuchs et al., 2011; Kim et al., 2011; Malczyk et al., 2013; Numaga-Tomita et al., 2016; Phelan, Shwe, Abramowitz, Birnbaumer, & Zheng, 2014; Smedlund, Birnbaumer, & Vazquez, 2015). Nevertheless, as elaborated in this review, the Drosophila TRP and TRPL channels are useful preparations for exploring mechanisms underlying fundamental properties of TRPC channels and their mode of activation and regulation in a rigorous manner. This is due to i) the highly developed genetic toolbox available for Drosophila, ii) Drosophila eye preparation being a native system expressing TRP channels, and iii) the striking similarity between Drosophila TRP and mammalian TRPCs, which enables translating findings from Drosophila to mammals.


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