Keywords

1 Introduction

The history of the transient receptor potential (TRP) ion channel family (Hardie 2011; Minke 2010; Montell 2011) dates back to the discovery of a spontaneously occurring mutant in a laboratory culture (Cosens and Manning 1969). Although the photoreceptors could generate a response, this decayed to baseline over a few seconds of continuous illumination rendering the flies effectively blind. Cosens and Manning called their mutant “A-type”; fortunately, Baruch Minke and colleagues, who studied this mutant extensively (Barash et al. 1988; Minke 1977, 1982; Minke et al. 1975), provided the more descriptive name, “transient receptor potential” or trp, which ultimately christened the entire gene family. When the trp gene was cloned, its sequence indicated a transmembrane (TM) protein with 8 TM helices (later revised to 6 plus a pore loop), with topology reminiscent of known receptor/transporter/channel proteins (Montell and Rubin 1989). However, there were no homologies to proteins in available databases and its function remained unknown. Intracellular recordings from trp mutant photoreceptors had suggested that the light-sensitive channels were normal (Minke 1982), apparently ruling out the possibility that the gene encoded the light-sensitive channel; instead, based on the ability of La3+ to mimic the trp phenotype, Minke and Selinger (1991) proposed it might represent a Ca2+ transporter required for refilling Ca2+ stores. Then, using whole-cell voltage-clamp recordings to determine ionic selectivity, Hardie and Minke (1992) showed that the primary defect in the trp mutant was a drastic reduction in the Ca2+ permeability of the light-sensitive channels themselves (Fig. 1). It was concluded that the normal light-sensitive current comprised two distinct conductances: one is highly Ca2+ selective and encoded by the trp gene and second is a channel responsible for the residual light-sensitive current in trp mutants. We now know the latter is encoded by a homologous gene: trp-like or trpl (Niemeyer et al. 1996; Phillips et al. 1992; Reuss et al. 1997). Drosophila TRP thus became the prototypical member of the TRP ion channel family and defines the “canonical” TRPC family, the first of which (TRPC1) was cloned by homology a few years later (Wes et al. 1995; Zhu et al. 1995).

Fig. 1
figure 1

The transient receptor potential phenotype. (a) In response to a prolonged stimulus (5 s step containing ~105 effective photons), wild-type photoreceptors show a rapid peak-plateau transition, relaxing to a steady state that is maintained indefinitely. Responses to brief test flashes (arrows) recover rapidly after the adapting light is turned off. In a trp mutant the response decays to baseline and sensitivity to test flashes is abolished, recovering only slowly in the dark (not shown). Shown here under whole-cell voltage-clamp conditions, this is the original transient receptor potential phenotype, which gives the TRP-channel family its name. The response decay is due to the failure of Ca2+-dependent inhibition of PLC, resulting in depletion of the entire pool of PIP2 in the microvilli (see Sect. 6). (b) Reversal potential (E rev) measurements. Responses to light flashes (arrows) in photoreceptors clamped at different holding potentials (in 10 mV steps) under bi-ionic conditions (130 mM Csi, 10 mM Cao). A large shift in E rev from ~35 mV in wild type to 0 mV in trp (expressing only TRPL channels) indicates a ~10-fold reduction in relative Ca2+ permeability. In trpl mutants (expressing only TRP channels), E rev is shifted to +42 mV. The wild-type response is a weighted response of both channels. A point mutation in the TRP channel (TRPD621G) virtually eliminates Ca2+ permeation, shifting E rev to −70 mV (Reprinted with modification from Reuss et al. (1997) with permission from Elsevier)

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This chapter primarily concerns the prototypical TRP channel and its homologue TRPL, which together mediate the light-sensitive current in Drosophila retinal photoreceptors (Niemeyer et al. 1996; Reuss et al. 1997). Related TRP channels that may mediate light-sensitive currents in atypical photoreceptor cells in other systems are also discussed at the end.

2 Gene and Protein Structure

The trp gene is located on the distal end of the third of Drosophila’s four chromosomes (cytogenetic map: 99C6-7). A single transcript with 15 exons encodes a 1,275 amino-acid protein with 6 transmembrane domains (S1–6) and a putative pore loop between S5 and S6 (Fig. 2). The trpl gene is on the second arm of chromosome 2 (46B2) and encodes a shorter peptide with 900 amino acids. Overall trpl shares ~40 % identity to trp; most of the divergence is within the C termini, and within the S1–6 the homology is much closer (~70 %). Within TM helices S3–S6, as first recognised by Phillips et al. (1992), there is also ~40 % identity with voltage-gated Ca2+ channels, although the positively charged voltage-sensing residues in S4 are replaced by polar residues. TRPs belong to the overarching superfamily of voltage and cyclic nucleotide-gated (CNG) ion channels, and like K channels and CNG channels, each gene encodes a subunit of a putative tetrameric ion channel. Although there is some evidence that TRP and TRPL may form heteromultimers when heterologously expressed (Xu et al. 1997), in the photoreceptors they are believed to form homo-tetramers in vivo (Katz et al. 2013; Reuss et al. 1997). Like other TRPC channels the N-termini of TRP and TRPL contain a coiled-coil region and 4 ankyrin repeats of unknown function, whilst the C-terminus includes the highly conserved “EWKFAR” motif of the “TRP box” found in several TRP subfamilies and one (TRP) or two (TRPL) calmodulin-binding sites. TRP has a conspicuously long C-terminal domain containing a proline-rich region, 8–9 peptide repeats of unknown function (Montell and Rubin 1989) and a PDZ-binding motif that binds to the scaffolding protein INAD (Peng et al. 2008; Shieh and Zhu 1996). The Drosophila genome includes a third TRPC channel—TRPγ (Xu et al. 2000). However, unlike TRP and TRPL, TRPγ is not particularly eye-enriched (Jors et al. 2006) and it seems doubtful whether it plays a role in phototransduction.

Fig. 2
figure 2

TRP and TRPL channels. (a) dTRP is a channel subunit with 6 transmembrane helices (S1–S6), with a pore loop between S5 and S6. The N-terminus contains a coiled-coil region and 4 ankyrin repeats. The “TRP domain” with the “EWKFAR” motif is a highly conserved region found in all TRPC channels. The C-terminus also contains a CaM binding site (CBS), a proline-rich region with 29 KP repeats, multiple repeats of a hydrophilic 8–9 peptide sequence DKDKKP(A/G)D and a PDZ-domain-binding motif required for binding to the INAD protein. (b) TRPL has similar N-terminal and transmembrane regions, but the C-terminal is distinct. The only recognised domains are two calmodulin-binding sites (CBS1 and CBS2). (c) Molecular model of the pore region of the TRP channel (2 subunits shown) based on the crystal structure of KcsA. An acidic residue (aspartate 621) in the pore loop is responsible for the high Ca2+ selectivity. (d) Like voltage-gated K+ channels and CNG channels, the TRP channel is believed to be a tetrameric assembly (probably homomeric) of 4 such subunits (a and b reprinted with modification from Hardie and Postma (2008) with permission from Elsevier; (c) Reprinted from Liu et al. (2007) with permission from the Journal of Neuroscience)

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3 Expression

Both TRP and TRPL are highly enriched in adult photoreceptors and were once considered photoreceptor-specific. However, genetic evidence also implicates them in olfaction (Stortkuhl et al. 1999) and Malphigian tubule function (Macpherson et al. 2005), whilst microarray data suggests limited expression in the brain (Flyatlas.org). TRPL, but not TRP, is also expressed in the larval eye, or Bolwig’s organ (Rosenbaum et al. 2011). In adult photoreceptors, both channel proteins are predominantly localised in the phototransduction compartment, a rod-like structure (rhabdomere) consisting of ~30,000 tightly packed microvilli (Fig. 3). Each microvillus probably contains about 20–30 TRP and 2–3 TRPL channels, representing channel densities of ~100/μm2 and ~10/μm2, respectively. TRPL, but not TRP, translocates reversibly from rhabdomere to cell body in response to illumination (Bahner et al. 2002), and a recent study also reported TRP and TRPL in the photoreceptor synaptic terminal where the channels may play a role in regulating transmitter release (Astorga et al. 2012).

Fig. 3
figure 3

Drosophila retina and phototransduction cascade. (a) Left: Diagram of ommatidium showing two (out of a total of eight) photoreceptors with their microvillar rhabdomeres. Light is focussed on the rhabdomere tips by the overlying lens (~16 μm in diameter). Each ommatidium is optically screened from its neighbours by pigment cells. The cross section (right) shows the geometrically precise array of rhabdomeres in the six R1–6 cells and a central photoreceptor R7 (a UV receptor). The electron micrograph shows a single rhabdomere (scale bar 500 nm) with tightly packed microvilli, each of which contain multiple copies of all major components of the transduction cascade. (b) Components of the transduction cascade shown approximately to scale in a schematic section of a “half” microvillus with its central actin filament. Photoisomerisation of rhodopsin (R) to metarhodopsin (M) activates Gq via GTP–GDP exchange (Step I), releasing the Gqα subunit; Gqα activates phospholipase C (PLC), generating InsP3 and DAG and a proton from PIP2 (Step II). Two classes of light-sensitive channels (TRP and TRPL) are activated, putatively by a combination of PIP2 depletion and acidification (Step III). DAG is also a potential precursor for polyunsaturated fatty acids (PUFAs). Exogenously applied PUFAs activate channels, but the lipase necessary for their generation in situ has not been demonstrated. Ca2+ influx, primarily through the TRP channels, feeds back at multiple targets, e.g., via PKC and CaM, thereby regulating, inter alia, the channels, PLC and M-Arr2 (arrestin). Ca2+ is extruded by the Na+/Ca2+ exchanger (NCX). Several components of the cascade, including TRP, protein kinase C (PKC) and PLC, are assembled into signalling complexes by the scaffolding protein, INAD, which may be linked to the central F-actin filament via the NINAC class III myosin. INAD contains 5 PDZ domains (1–5) (Reprinted with modification from Yau and Hardie (2009) with permission of Elsevier)

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During pupal development, which lasts ~100 h, TRP protein is first detected by immunocytochemistry at ~45 h (Satoh et al. 2005). However, functional TRP-channel activity is only detectable from ~82 h, whilst functional TRPL channels are first detected even later at ~90 h (Hardie et al. 1993). Little is known about the secretory pathway and targeting; however, TRP trafficking requires both the Rab GTPase Rab11 (Satoh et al. 2005) and a novel chaperone protein (XPORT) both of which are required for successful targeting of both TRP and rhodopsin to the rhabdomeric membrane (Rosenbaum et al. 2011).

Despite many attempts, it has proved very difficult to express TRP heterologously. In the few apparently successful attempts (Gillo et al. 1996; Vaca et al. 1994; Xu et al. 1997), the reported biophysical properties did not closely match those of the native trp-dependent current. Any doubts this might raise over the identity of TRP as the light-sensitive channel were dispelled with the identification of a unique aspartate residue within the pore loop as the major determinant of Ca2+ permeation (Liu et al. 2007). Targeted mutagenesis of this residue resulted in systematic alteration of pore properties in vivo demonstrating that TRP indeed forms a pore-forming subunit of the Drosophila light-sensitive channel (see Sect. 5.1).

By contrast TRPL has been successfully expressed in many expression systems (Gillo et al. 1996; Hambrecht et al. 2000; Hardie et al. 1997; Harteneck et al. 1995; Hu et al. 1994; Lan et al. 1996). Its biophysical properties are essentially indistinguishable from those of the native TRPL-dependent current isolated in trp mutants (Chyb et al. 1999; Hardie et al. 1997), with the exception that in many expression systems it is constitutively active (for recent discussion, see Lev et al. 2012).

4 Interacting Proteins

4.1 Scaffolding Protein INAD

Along with other key components of the cascade, TRP channels are organised into multimolecular signalling complexes by the “inactivation no afterpotential D” (INAD) protein (Fig. 3), a scaffolding protein with five PDZ domains (Chevesich et al. 1997; Shieh and Niemeyer 1995; Shieh and Zhu 1996; Tsunoda et al. 1997). PDZ domains typically bind to a terminal three amino-acid motif on the target protein; however, the interaction of TRP with INAD appears to involve up to 14 amino acids in the C-terminal (Peng et al. 2008). In vitro evidence suggests the interaction may be “bidentate”, with PDZ3 forming a classical association with the terminal 3 amino acids (GWL) of TRP and the upstream residues interacting with the PDZ4/5 supramodule (Liu et al. 2011). As well as TRP, INAD binds stoichiometrically (1:1) with PLC and PKC (Huber et al. 1996a; Li and Montell 2000; Tsunoda et al. 1997). INAD has also been reported to interact with NINAC, a class III myosin that may link the complex to the actin cytoskeleton (Hicks et al. 1996; Wes et al. 1999): calmodulin; retinophilin (Mecklenburg et al. 2010) and the immunophilin FKBP59 (Goel et al. 2001; Huber 2001; Montell 1999). Although TRPL has also been reported to bind INAD (Xu et al. 1998), this has been questioned, and a quantitative study suggests that at most only a small fraction of TRPL protein could associate with INAD (Paulsen et al. 2000). INAD can form homomultimers in vitro (Xu et al.1998), and together with other potential linkages (e.g., via TRP subunits which form tetramers), it is possible that extended complexes are formed.

Molecular scaffolds like the INAD complex promote signalling specificity and might facilitate rapid kinetics by minimising diffusional delays. In apparent support of this, null inaD mutants have reduced sensitivity and slow responses (Scott and Zuker 1998); however, these phenotypes could also simply reflect the greatly reduced levels of microvillar PLC found in inaD mutants (Tsunoda et al. 2001). Whether or not the integrity of the scaffolding complex is critical for rapid excitation, INAD is required to maintain high concentrations of TRP, PLC and PKC in the rhabdomere in the correct stoichiometry (Cook et al. 2000; Huber et al. 1996a). In fact, TRP and INAD are mutually required for the long-term stability of the scaffolding complex; thus TRP protein disappears from the rhabdomere in inaD mutants over a period of days, whilst INAD protein is reciprocally destabilised in trp mutants (Li and Montell 2000; Tsunoda et al. 1997).

Structural studies of the fourth and fifth PDZ domains in INAD show that PDZ4/5 can exist in two redox states due to the reversible light and PKC-dependent formation of a Cys-Cys disulfide bridge which disrupts the binding site—possibly for TRP or PLC (Liu et al. 2011; Mishra et al. 2007). Mutants in which PDZ4/5 is locked in one or the other state show specific defects in response to inactivation and/or activation. This raises the possibility that rather than acting as just a passive scaffold, INAD may regulate signal transduction on a millisecond timescale through dynamic switching between conformational states (Liu et al. 2011; Mishra et al. 2007).

4.2 INAF

The inaF mutant has a response decay phenotype very similar to trp (Li et al. 1999). When first cloned the inaF gene was considered novel without vertebrate homologues; however, a second transcript was later identified with a transmembrane motif that is conserved from flies to humans, but of unknown function (Cheng and Nash 2007). Using an HA-tagged inaF transgene, INAF was found to localise to the rhabdomeres and to co-immunoprecipitate with TRP (Cheng and Nash 2007). In null inaF mutants TRP protein levels are reduced to ~10 %, but the residual protein still localises to the rhabdomeres (Li et al. 1999). Despite this residual protein, the inaF phenotype is as severe as in null trp mutants, perhaps implicating INAF as an auxiliary subunit required for TRP-channel function, (Li et al. 1999).

4.3 Calmodulin

Both TRP and TRPL are calmodulin (CaM)-binding proteins, and both TRP and TRPL are potently regulated by Ca2+ (see below); however, surprisingly little is known of CaM’s role in this regulation.

TRPL has two CaM-binding sites (CBS1 and CBS2) in its C-terminus, but their roles are still unclear. In vitro studies give somewhat conflicting results: Warr and Kelly (1996) reported that CBS1 is conventional, binding CaM in a Ca2+-dependent fashion, whilst CBS2 binds the Ca2+-free form of CaM, with dissociation occurring at high (>10 μM) Ca2+ concentrations. However, Trost et al. (1999) reported that both sites bind CaM in the presence of Ca2+, but with slightly different Ca2+ dependencies (50 % binding at 100 nM and 3.3 μM Ca2+, respectively, for CBS1 and CBS2). TRPL channels are strongly inhibited by Ca2+ with an IC50 of 1 μM or lower (Obukhov et al. 1998; Parnas et al. 2007; Reuss et al. 1997); however, CaM’s role, if any, in this regulation is unclear. Scott et al. (1997) reported that Ca2+-dependent inhibition of TRPL channel activity in vivo was impaired in trpl mutants lacking either CBS1 or CBS2; however, our lab was unable to reproduce this result using the same transgenic flies (Hardie R.C. unpublished). When expressed in Sf9 cells, the inhibitory action of Ca2+ on TRPL was unaffected by the CaM inhibitor calmidazolium (Obukhov et al. 1998). There are, however, reports of facilitation of TRPL channels by Ca2+-CaM in heterologous expression systems (Lan et al. 1998; Trost et al. 1999).

The TRP protein has one CBS in the C-terminus, and peptide fragments from this region bind CaM in a Ca2+-dependent manner in vitro (Chevesich et al. 1997). TRP channels are both positively (EC50 300 nM) and negatively (IC50 ~ 1 μM) regulated by Ca2+ (Chu et al. 2013a; Gu et al. 2005), but again, whether CaM is involved in this regulation is not known.

4.4 Phosphorylation

Both TRP and TRPL proteins have multiple phosphorylation sites. TRP is a target for PKC, which is also a component of the INAD scaffolding complex (Huber et al. 1996b, 1998; Liu et al. 2000). Popescu et al. (2006) identified Ser982 on the TRP C-terminus as an in vitro target for PKC and found a mild slow-deactivation phenotype (albeit rather less severe than in a PKC null mutant) in Ser to Ala mutants of this site. More recently, 21 phosphorylation sites on the native TRP protein were identified by mass spectrometry, mainly located in the C-terminus (Voolstra et al. 2010). Several of these, including Ser982, were phosphorylated in a light-dependent manner, but surprisingly none were found to be compromised in a PKC null mutant, indicating either that they were not PKC targets or that they can be redundantly phosphorylated by additional kinases. Although most sites were phosphorylated in response to light, one in particular (Ser936) was dephosphorylated in response to illumination (Voolstra et al. 2010). Apart from the mild phenotype in TRPS982A mutants, no function has been attributed to any of these sites.

In vitro studies of recombinant TRPL peptide fragments found that CaM binding to CBS1 was prevented by PKA-dependent phosphorylation, whilst phosphorylation by PKA was inhibited by prior PKC-dependent phosphorylation (Warr and Kelly 1996). Again no function has been attributed to this regulation. Recently, mass spectrometry identified eight C-terminal, light-dependent phosphorylation sites in TRPL. Mutation of these sites did not affect the electrophysiological response to light, but led to reduced TRPL content and partial mislocalisation out of the rhabdomere (Cerny et al. 2013).

4.5 Miscellaneous Interacting Proteins

At least three further proteins have been reported to co-immunoprecipitate with TRP and/or TRPL, although a direct interaction has not been demonstrated.

  1. 1.

    FKBP59, the Drosophila homologue of the human FK506-binding protein, was identified as an INAD binding partner in a yeast-2-hybrid screen and also found to co-immunoprecipitate with TRPL (Goel et al. 2001). The function of this interaction has not been studied in vivo, but when co-expressed in Sf9 cells, FKBP59 induced a graded inhibition of TRPL activity in fura-2 Ca2+ influx assays.

  2. 2.

    Moesin, which is the sole member of the ezrin-radixin-moesin (ERM) family in Drosophila, co-immunopreciptates with both TRP and TRPL (Chorna-Ornan et al. 2005). ERM proteins promote actin-membrane interactions (Fehon et al. 2010) and are required for the development and maintenance of the microvilli (Karagiosis and Ready 2004). Upon illumination and PIP2 depletion, moesin becomes dephosphorylated, dissociates from TRP and/or TRPL and migrates reversibly from the base of the rhabdomere to cytosolic regions (Chorna-Ornan et al. 2005; Sengupta et al. 2013).

  3. 3.

    TRP (but not TRPL) interacts with XPORT, which is a resident ER protein in the secretory pathway and which was recently identified as chaperone for both TRP and rhodopsin (Rosenbaum et al. 2011).

5 Biophysical Description of Channel Function, Permeation and Gating

5.1 Ionic Selectivity

The biophysical properties of the native TRP and TRPL conductances in vivo have been characterised in null trp and trpl mutants to isolate the respective currents (e.g., Reuss et al. 1997) (Fig. 1). Both channels permeate a broad range of monovalent and divalent ions including Ca2+, Mg2+, Ba2+, Sr2+ and Mn2+ (Table 1). TRP and TRPL differ most notably in their selectivity for divalent ions: TRP channels are highly selective for Ca2+ (P Ca:P Cs >50:1), and of the entire TRP superfamily, only the truly Ca2+-selective TRPV5 and TRPV6 have a higher selectivity. TRPL has a Ca2+ selectivity (P Ca:P Cs ~ 4:1) more typical of most TRP channels, including the broader TRPC subfamily (Hardie and Minke 1992; Liu et al. 2007; Reuss et al. 1997).

Table 1 Relative permeabilities of TRP and TRPL channels determined under bi-ionic conditions from reversal potential measurements of the light-induced currents
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The high Ca2+ selectivity of TRP has been attributed to a single negatively charged aspartate residue (Asp621) located in the pore loop. Neutralising Asp621 (by mutation to glycine or asparagine) eliminates Ca2+ permeation as well as the block by Mg2+ and ruthenium red leaving a monovalent cation channel (Liu et al. 2007). Increasing the side-chain length but maintaining the positive charge (Asp > Glu) barely affected Ca2+ selectivity but decreased the effective pore size, suggesting the residue also resides at the narrowest part of the channel. By analogy with other Ca2+-selective channels, it seems likely that four such Asp621 residues (one from each subunit) coordinate a Ca2+-binding site in the mouth of the pore (Figs. 1 and 3). A conserved charged residue (Glu) in the equivalent position was also found to be responsible for Ca2+ selectivity in mammalian TRPC3 (Poteser et al. 2011).

5.2 Single-Channel Properties

Under physiological conditions noise analysis of native currents in photoreceptors suggests single-channel conductances of ~8 pS for TRP and 35 pS for TRPL in the presence of divalent cations (1.5 mM Cao and 4 mM Mgo) or ~30 pS (TRP) and 68 pS (TRPL) under divalent-free conditions (Hardie et al. 1997; Henderson et al. 2000; Reuss et al. 1997). In excised patch single-channel recordings from rhabdomeric membrane, values of 57 pS (TRP) and 49 pS (TRPL) have been reported under divalent-free conditions (Delgado and Bacigalupo 2009). When expressed in S2 cells, TRPL had a single-channel conductance of 110 pS in the presence of 4 mM Mg and 0 Ca (Parnas et al. 2007).

Based on permeability to a series of organic cations, the pore diameter of TRP was estimated at ~8 Å (Liu et al. 2007). Pore diameter has not been systematically measured for TRPL, although it was reported to be essentially impermeant to tetraethylamine and N-methyl-d-glucamine (Parnas et al. 2009a).

Open times of both channels are brief: TRPL openings are characterised by two time constants (~0.1 ms and 0.5–2 ms) both in single-channel recordings in S2 cells and in noise analysis from spontaneously active TRPL channels in trp mutants (Hardie and Minke 1994; Hardie et al. 1997; Parnas et al. 2007). Based on power spectra of native channels, TRP-channel kinetics are even faster, and only a single time constant of ~0.5 ms can be resolved (Raghu et al. 2000b). In either case the very brief channel openings mean that physiological response to illumination should not be limited by channel kinetics.

5.3 Voltage Dependence and Divalent Ion Open-Channel Block

Under physiological conditions, in the presence of divalent cations, TRPL channels show pronounced outward rectification, whilst TRP channels display complex dual inward and outward rectifying characteristics. In both cases voltage-dependent divalent ion open-channel block appears to be responsible.

5.3.1 TRP Channels

For TRP channels, the dual inward/outward rectification is abolished and the effective single-channel conductance increased 10-fold under divalent-free conditions (Hardie and Mojet 1995). Using Mg2+ as the blocking ion, IC50 (under Ca2+ free conditions) was reported as ~280 μM. The dual rectification was interpreted as voltage-dependent relief of flickering open-channel block (OCB) by divalent ions with both depolarisation and hyperpolarisation sufficient to drive the blocking ions from the pore. Under physiological conditions the voltage-dependent block is relatively weak around resting potential (−70 mV) but intensifies as the cell depolarises. This means that the effective single-channel conductance should decrease as the cell depolarises in response to light, potentially representing an economical mechanism for light adaptation (Hardie and Mojet 1995).

5.3.2 TRPL channels

The I–V relation of the TRPL channel is strongly outwardly rectifying in physiological solutions but becomes essentially linear under divalent-free conditions. The outward rectification under physiological conditions is thus interpreted as voltage-dependent relief of OCB (Parnas et al. 2007). In many cases of OCB, the kinetics of block and unblock are too fast to be resolved resulting in a reduction in effective single-channel conductance (flickering block) as appears to be the case for the TRP channel. However, in a detailed single-channel analysis of heterologously expressed TRPL channels, removal of divalent ions or depolarisation only increased open probability and open time without affecting single-channel conductance (Parnas et al. 2007). The divalent ion OCB described by Parnas et al. (2007) had an IC50 of 163 nM for Cao at −70 mV and ~2 mM for Mg2+.

5.4 Pharmacology

Like many Ca2+ channels and several mammalian TRP channels, TRP, but not TRPL, channels are completely blocked by low μM levels of La3+ and ruthenium red (Liu et al. 2007). The highest affinity blocker reported for TRPL is cinnamyl-dihydroxy-cyanocinnamate, with an IC50 of 1 μM (Chyb et al. 1999). A variety of compounds, which are activators of some of the thermo-TRPs, inhibit TRPL channels at much higher concentrations, including carvacrol (IC50 357 μM), thymol (1 mM), eugenol (3 mM), cinnamaldehyde (2 mM) and menthol (1.8 mM) (Parnas et al. 2009b). We recently found that a range of amphiphilic weak bases were all reversible inhibitors of both TRP and TRPL activity. Although the potency varied widely (IC50 for TRPL: trifluoperazine 5 μM, chlorpromazine 25 μM, imipramine 100 μM, procaine 5 mM) after correction for partitioning and pKa values (in an attempt to predict concentration in the lipid membrane), the IC50 values converged near a common value of ~5 mM, suggesting their action could be attributed to the effect of the weak bases on physico-chemical properties of the lipid bilayer (Hardie and Franze 2012).

6 Physiological Functions

The primary function of the Drosophila TRP and TRPL channels is clear: TRP is the major light-sensitive channel in the photoreceptors of the retina. It accounts for up to 95 % of the light-sensitive current, the remainder carried by its homologue TRPL (Reuss et al. 1997).

6.1 Mechanism of Activation

Phototransduction in Drosophila has long been recognised as a G protein-coupled PLC-based cascade (Bloomquist et al. 1988; Pak 1995). All the essential elements of the cascade including the channels are localised within ~30,000 microvilli together forming the light-guiding rhabdomere (Fig. 3). As in other photoreceptors the cascade is initiated by absorption of a photon by a chromophore (3-OH 11-cis retinaldehyde) covalently attached to rhodopsin (R). Photoisomerisation of 11-cis retinal to the all-trans configuration converts R to the active metarhodopsin state (M), which catalytically activates a heterotrimeric Gq protein releasing the active Gqα subunit (Scott et al. 1995). A handful, perhaps 5–10, Gqα are released by each activated M within the same microvillus during a brief but variable latency period (15–100 ms) and diffuse in the plane of the membrane before binding to and activating a PLC molecule (Hardie et al. 2002). Each of the 5–10 PLC molecules thus activated then hydrolyses PIP2 at a rate of ~1,000 molecules per second yielding InsP3, DAG and a proton. The final electrical response in response to absorption of a single photon is a quantum bump, ~10 pA in amplitude, probably representing the opening of most of the 20 or so TRP channels in a single microvillus. Under voltage-clamp conditions, the output of the cell as a whole represents the summation of the quantum bumps independently generated over the 30,000 microvilli (see Hardie 2012 for recent detailed review).

Which consequences of PLC activity are responsible for channel activation remains debated. Most evidence suggests that InsP3-induced Ca2+ release plays no role (Hardie 1995; Hardie and Raghu 1998) with phototransduction and adaptation appearing completely normal in InsP3 receptor mutants (Acharya et al. 1997; Raghu et al. 2000a).

6.1.1 Diacylglycerol and Polyunsaturated Fatty Acids

TRP and TRPL channels are constitutively active in mutants of diacylglycerol kinase (DGK), encoded by the rdgA gene (Raghu et al. 2000b). Because DGK metabolises DAG to phosphatidic acid (PA), this is consistent with the notion that the constitutive activity in rdgA mutants is due to the build-up of DAG by basal PLC activity (Raghu et al. 2000b). Genetic interactions between rdgA and norpA (encoding PLC) or Gαq (Gqα subunit) also support an excitatory role for DAG, namely, in hypomorphic Gαq and norpA mutants, responses to light are greatly reduced due to diminished PLC activity, but when combined with the rdgA mutation (rdgA,norpA or rdgA;Gαq double mutants), the residual responses are greatly facilitated (Hardie et al. 2002).

Since DAG activates several mammalian TRPC channels (Hofmann et al. 1999), DAG seems a plausible candidate for the excitatory second messenger; however, there are problems with this suggestion (Katz and Minke 2009; Raghu 2006; Raghu and Hardie 2009). First, conversion of DAG to PA by DGK is the first step in PIP2 resynthesis, and hence phenotypes reported in rdgA might also reflect reduced PIP2 levels (Garcia-Murillas et al. 2006; Raghu and Hardie 2009). In addition, PA potently facilitates PI(4)P 5-kinase (Cockcroft 2009; Jenkins et al. 1994), so the final stage of PIP2 resynthesis may also be compromised in rdgA mutants. Second, DGK appears to immunolocalise predominantly to the endoplasmic reticulum and not the microvilli where other components of transduction are localised (Masai et al. 1997). Third, attempts to activate TRP and TRPL channels with exogenous DAG have, in general, been unsuccessful (but see Delgado and Bacigalupo 2009).

By contrast polyunsaturated fatty acids (PUFAs), such as arachidonic and linolenic acid (which, in principle, might be released from DAG by a DAG lipase), robustly activate native TRP and TRPL channels as well as heterologously expressed TRPL channels (Chyb et al. 1999; Estacion et al. 2001; Parnas et al. 2009a) with an EC50 of ~10 μM. Apparent genetic evidence for PUFAs as the endogenous excitatory messenger came from mutants of a DAG lipase gene, inaE, which have greatly reduced sensitivity to light (Leung et al. 2008). However, the inaE DAG lipase is of the sn-1 class, which releases monoacylglycerols rather than PUFAs from DAG; whilst the INAE protein immunolocalises to the cell body with, at most, isolated traces in the rhabdomeres (Leung et al. 2008). There is also no evidence that light results in generation of PUFAs in the photoreceptors. As discussed below it is possible that PUFAs may be acting as surrogate agonists, mimicking the effects of PIP2 hydrolysis on the physico-chemical properties of the lipid bilayer.

6.1.2 PIP2 Depletion, Protons, and Bilayer Mechanics

In addition to generating DAG and InsP3, hydrolysis of PIP2 by PLC has two further actions: (1) it depletes PIP2 and (2) it releases a proton. A recent study indicated that these two neglected consequences of PLC activity can act combinatorially to activate the channels (Huang et al. 2010). Thus, following depletion of PIP2 in the photoreceptors, both TRP and TRPL channels could be rapidly and reversibly activated by protonophores, whilst heterologously expressed TRPL channels in S2 cells could be activated by acidification of the cytosolic surface of inside-out patches (Fig. 4). This indicates that a combination of PIP2 depletion and acidification is sufficient to activate the light-sensitive channels (Huang et al. 2010).

Fig. 4
figure 4

Activation by protons and PIP 2 depletion. (a) Upon illumination (at time zero), there is a rapid acidification of the photoreceptor (measured with the fluorescent pH indicator, HPTS, loaded via whole-cell recording electrode: the signal is significantly faster when imaged from the rhabdomere than from the whole cell). (b) Under control conditions (left) brief application of the protonophore 2,4 dinitrophenol (DNP, 100 μM) activates no channels (whole-cell recording) in a trp mutant. However, after PIP2 depletion induced by a 5 s light pulse, the same dose of DNP rapidly activates the light-sensitive channels. (c) Patch-clamp recording from an excised inside-out patch containing TRPL channels expressed in Drosophila S2 cells. Acidification of the bath (cytosolic surface of patch) rapidly and reversibly activated the channels. (d) pH dependence of channel activation (Reprinted with modification from Huang et al. (2010) with permission from Elsevier)

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These results appear to suggest that the channels are activated by protons and that PIP2 binding to the channel inhibits this, either allosterically or by masking a protonatable site. Arguing against this however, PIP2 applied to the cytosolic surface of inside-out patches actually activated TRPL channels expressed in S2 cells (Huang et al. 2010)—although the opposite result was reported in Sf9 cells (Estacion et al. 2001). An alternative possibility is that by removing its bulky and charged headgroup, hydrolysis of PIP2 results in a change in the mechanical properties of the lipid bilayer, as has been suggested, albeit controversially, for the PLC-mediated activation of heterologously expressed TRPC6 (Spassova et al. 2006). A similar idea was also championed by Parnas et al. (2009a) who suggested that lipid activators such as PUFAs might exert their effect by modulating channel interactions with the lipid bilayer rather than on the channel per se. In support of this they found that heterologously expressed TRPL channels were influenced by other manipulations expected to alter bilayer properties including facilitation by osmotic stretch and suppression by GsMTx-4 toxin, a mechano-sensitive ion channel blocker which acts on the channel/lipid boundary (Suchyna et al. 2000). They also found that the divalent ion-dependent outward rectification of TRPL channels was linearised by PUFA application and proposed that activation of channels by such lipid-mediated interaction was achieved by removal of open-channel block (Parnas et al. 2009a).

In support of a “photomechanical” mechanism, photoreceptors were recently shown to contract in response to light (Hardie and Franze 2012). The contractions, measured using atomic force microscopy on isolated retina, were faster than the electrical response (latency <5 ms); were strictly dependent upon PLC activity; and in the absence of Ca2+ influx (which normally inhibits PLC) were saturated at intensities equivalent to ~1 absorbed photon per microvillus. The contractions seem most parsimoniously explained by the decrease in membrane area due to hydrolysis of the large headgroup of PIP2 from the inner leaflet, thereby increasing membrane tension and resulting in constriction of the microvilli. Furthermore, light responses, whether mediated by TRP or TRPL channels, were reversibly increased or suppressed by hypo- and hyper-osmotic solutions, respectively, indicating that the native channels were sensitive to membrane tension (Fig. 5). To ask whether light-induced hydrolysis of PIP2 generated sufficient mechanical force to activate ion channels, a known mechano-sensitive channel (gramicidin) was incorporated into the membranes of trpl;trp double mutant photoreceptors lacking all native light-sensitive channels. Remarkably, such photoreceptors now responded to light by increased activity of the ectopic gramicidin channels with similar intensity dependence to the measured contractions (Hardie and Franze 2012).

Fig. 5
figure 5

PIP 2 depletion and photomechanical response. (a) The bulky, charged headgroup of PIP2 is cleaved by PLC leaving DAG, with its much smaller footprint, in the membrane. Cartoon cross-sections of a microvillus indicate how removal of the inositol headgroup (blue) from the inner leaflet would decrease crowding, thereby increasing membrane tension, with the result that the microvillar diameter may contract. (b) (1) Contractions of the photoreceptors measured by AFM (lower traces) to moderate intensity flashes (200–800 effective photons); upper traces show electrical responses recorded to identical stimuli. (2) Family of contractions measured to 5 ms flashes of increasing intensity: latency to brightest flashes is less than 5 ms (cf ~ 6 ms for electrical response). (3) Response intensity function for contractions (nm) and electrical response (mV) mediated by TRP channels recorded under current clamp conditions. (c) (i) Left: Whole-cell voltage-clamp light responses in a trp mutant to a series of brief flashes are rapidly and reversibly facilitated by hypo-osmotic solution (200 mOsm). Right: Representative superimposed flash responses mediated by both TRPL channels (in trp mutant) and TRP channels (in trpl mutant) are suppressed by hyper-osmotic (400 mOsm) and facilitated by hypo-osmotic (200 mOsm) solutions. Bar graph (ii) shows normalised responses for wild type (wt), trp and trpl, as well as wt in Ca2+-free solution to exclude indirect effect by change in Ca2+ (Reprinted with modification from Hardie and Franze (2012) with permission from Science)

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Given the dual requirement for PIP2 depletion and protonophores, a working hypothesis would be that the altered physical membrane environment following PIP2 hydrolysis is energetically favourable for a conformational state of the channel that can be directly activated by protonation.

6.2 Ca2+ Influx

As well as mediating the cell’s electrical response to light, TRP and to some extent TRPL channels are responsible for massive Ca2+ influx during the light response. Measurements of fractional Ca2+ currents indicate that ~30 % of the light-sensitive current is carried by Ca2+ ions, broadly in line with GHK predictions (Chu et al. 2013b). As a result, the concentration of Ca2+ reached transiently within each microvillus during a quantum bump probably exceeds 1 mM, saturating even the lowest affinity Ca2+ indicators (Oberwinkler and Stavenga 2000; Postma et al. 1999). During maintained illumination in a light-adapted state, this relaxes to a global steady-state concentration of around 5–10 μM due to the action of a potent Na+/Ca2+ exchanger.

The Ca2+ influx, particularly via the dominant TRP channels, plays essential positive and negative feedback roles in regulating the gain and kinetics of the light response including most aspects of light adaptation (Gu et al. 2005; Hardie 1991; Henderson et al. 2000; Reuss et al. 1997). The importance of Ca2+ influx is shown by removing extracellular Ca2+ from the bath, in which case both rising and falling phases of the response become at least tenfold slower, and light adaptation is abolished. The positive feedback is primarily, if not exclusively, reflected in facilitation of TRP-channel activity, whilst both TRP and TRPL channels are inhibited by Ca2+ (Reuss et al. 1997). Although Ca2+ influx influences many molecular targets, both positive and negative feedback effects may be mediated directly on the channels themselves (Gu et al. 2005; Hardie 1995). Using the transmembrane Na+ gradient to control cytosolic Ca2+ via the Na+/Ca2+ exchanger equilibrium, the IC50 for both TRP and TRPL channel inhibition was estimated at ~ 1μM (Gu et al. 2005), whilst TRP, but not TRPL, channel activity, induced by exogenous agonist (PUFAs), was facilitated by Ca2+ with an EC50 of ~ 300 nM (Chu et al. 2013a).

Other Ca2+-dependent targets modulated by influx via the TRP and TRPL channels include Ca2+-CaM-dependent acceleration of arrestin binding to activated metarhodopsin (Liu et al. 2008) and Ca2+-dependent inhibition of PLC, which appears to be mediated via PKC—possibly indirectly via phosphorylation of INAD (Gu et al. 2005). In addition, Ca2+ at low (submicromolar) concentrations can facilitate PLC (Hardie 2005; Katz and Minke 2012; Running Deer et al. 1995), although it is unclear whether this is significant under physiological conditions. There are also at least two Ca2+-dependent steps in the visual pigment cycle: CaMKII-dependent phosphorylation of Arr2 and Ca-CaM-dependent dephosphorylation of rhodopsin by rhodopsin phosphatase (rdgC gene) (Byk et al. 1993; Steele et al. 1992; Vinos et al. 1997). Although neither appear to contribute directly to the electrophysiological response kinetics, both are required for normal pigment recycling and long-term cell survival (see Sect. 8).

7 Channel Mutant Phenotypes

7.1 trp Phenotype

The explanation for the classical transient receptor potential phenotype, namely, the decay of the response to baseline during continuous illumination (Fig. 1), has been controversial. Originally it was interpreted as the depletion of InsP3-sensitive Ca2+ stores due to reduced Ca2+ influx (Cook and Minke 1999; Minke and Selinger 1991) and later as Ca2+-dependent inhibition of the TRPL channels mediating the residual response (Scott et al. 1997). However, subsequent studies failed to support a role of intracellular Ca2+ stores in phototransduction (Acharya et al. 1997; Hardie 1996; Raghu et al. 2000a; Ranganathan et al. 1994), whilst the trp phenotype was found to be accentuated in the absence of Ca2+ influx (Cook and Minke 1999; Hardie et al. 2001). What then causes the phenotype that effectively christened the TRP ion channel family? An answer came using a genetically targeted PIP2-sensitive ion channel (Kir2.1) to monitor endogenous PIP2 levels in vivo. This showed that the decay of the response in trp mutants was strictly paralleled by the rapid depletion of PIP2, whilst the recovery of sensitivity in the dark followed the time course of PIP2 resynthesis (Hardie et al. 2001, 2004). The underlying cause for PIP2 depletion and response decay appears to be the failure of Ca2+ and PKC-dependent inhibition of PLC, which is normally dependent upon Ca2+ influx via the TRP channels (Gu et al. 2005; Hardie et al. 2001). Without this inhibition, PLC activity is sufficient to hydrolyse all PIP2 in the rhabdomere within as little as a second of relatively modest illumination (Hardie et al. 2001, 2004).

The absence of the major Ca2+ influx channel, and the resultant PIP2 depletion, has many secondary phenotypic consequences including lack of light adaptation, lack of positive feedback, reduced single-photon responses (quantum bumps) as well as light-dependent retinal degeneration (see below).

7.2 trpl Phenotype

The TRP channel dominates the wild-type light response, and in whole-cell voltage-clamp recordings there is little obvious phenotype in trpl null mutants beyond predictable changes in permeation properties and pharmacology (Niemeyer et al. 1996; Reuss et al. 1997). However, studies under more physiological conditions, using either the electroretinogram (ERG) or the intracellular voltage recordings, revealed further subtle phenotypes. These include a reduced plateau potential, high-frequency (~100 Hz) oscillations superimposed on the response and an impaired ability to light adapt to very dim background lights (Bahner et al. 2002; Leung et al. 2000). With the possible exception of the reduced ability to light adapt, it remains unclear how these phenotypes relate to the known properties of the two channels. The oscillations in the trpl mutant ERG are probably synaptic in origin and may reflect a role of TRPL channels in synaptic transmission (Astorga et al. 2012).

8 Role in Hereditary and Acquired Diseases

TRP channels are the primary route for Ca2+ influx in the photoreceptor and play a major role in Ca2+ homeostasis, balanced by an NCX Na+/Ca2+ exchanger which is the major mechanism for Ca2+ extrusion (Wang et al. 2005b). A variety of retinal pathologies have been attributed both to reduced Ca2+ influx in mutants where TRP-channel activity is reduced or eliminated and to excessive Ca2++ influx under conditions where channels are constitutively activated or fail to inactivate on cessation of the light stimulus.

Mutants in upstream components of the cascade, e.g., Gαq or PLC, result in failure to activate channels and typically undergo light-dependent retinal degeneration over a course of one to two weeks. This is believed to result from failure of one or more Ca2+-dependent steps in the visual pigment cycle resulting in the build-up of potentially toxic complexes consisting of arrestin (Arr2) bound to phosphorylated M (MPP-Arr2). Thus, the pigment cycle normally involves photoisomerisation of R to M, whose activity is terminated by Ca2+-dependent binding to Arr2 (Dolph et al. 1993; Liu et al. 2008). At the same time M becomes phosphorylated by Rh kinase. Although Rh kinase is probably not Ca2+-dependent per se, phosphorylated rhodopsin is dephosphorylated by a Ca2+-CaM-dependent phosphatase encoded by the rdgC gene so that rhodopsin becomes hyperphosphorylated in the absence of Ca2+ (Lee and Montell 2001; Steele et al. 1992; Vinos et al. 1997). Arr2 itself is also rapidly phosphorylated by CaMKII (Matsumoto et al. 1994). This is important for two reasons: first, unphosphorylated Arr2-MPP is a target for clathrin-mediated endocytosis which is believed to trigger apoptosis (Kiselev et al. 2000) and, second, Arr2 can only dissociate from R once Arr2 has been phosphorylated (Alloway and Dolph 1999). Red light can photoreisomerise M back to R at any point during the cycle and thus also prevents build-up of toxic MPP-Arr2.

trp mutants themselves also undergo retinal degeneration. There is evidence suggesting that defects in the pigment cycle contribute to the degeneration (Lee et al. 2013; Wang et al. 2005a); however, recently evidence was found for an additional mechanism, namely, the profound PIP2 depletion that occurs when Ca2+ influx is compromised (Sengupta et al. 2013). MPP-Arr2-mediated degeneration can be simply prevented by rearing flies under red light (which reconverts M to R); this rescues degeneration in norpA and rdgC, but not in trp. Instead degeneration under red light was rescued by genetic elimination of PLC (in a norpA;trp double mutant), whilst the intensity dependence of degeneration quantitatively matched that of PIP2 depletion. PIP2 depletion and degeneration were also associated with depolymerisation of the actin cytoskeleton of the microvilli, possibly triggered by dephosphorylation of moesin (Sengupta et al. 2013).

Excessive Ca2+ influx is widely implicated in excito-toxicity, and, not surprisingly, overactivity of the photoreceptor TRP channels also triggers retinal degeneration. Again this can be due to defects in upstream signalling or in the TRP channel itself. For example, light-dependent retinal degeneration is found in arr2 mutants, attributable to persistent channel activity due to failure to inactivate M (Dolph et al. 1993). Particularly severe degeneration occurs in DAG kinase (rdgA) mutants, which are characterised by constitutive TRP and TRPL channel activity (Raghu et al. 2000b). However, the most direct and striking evidence implicating the cytotoxic effect of Ca2+ influx via TRP channels comes from the dominant Trp 365 mutant, which has a point mutation (Phe550Ile) in S5 that results in a constitutively active channel and massive retinal degeneration even in the dark (Hong et al. 2002; Yoon et al. 2000). Interestingly this aromatic residue is widely conserved amongst the TRP family, and the equivalent mutation in the yeast TRPY1 channel is also a gain-of-function mutation with increased constitutive activity (Su et al. 2007).

9 Other “Photosensitive” TRP Channels

9.1 ipRGCs

Vertebrate rods and cones are the so-called ciliary photoreceptors which diverged from the microvillar photoreceptors characteristic of Drosophila and many other invertebrates at least 550 million years ago. It had long been assumed that rods and cones, which signal using cGMP-gated channels, were the only photoreceptors in vertebrate eyes. However, recently it has become recognised that there are additional classes of light-sensing cell in most if not all vertebrate retinae (Berson 2003; Do and Yau 2010; Fu et al. 2005; Hankins et al. 2008). The best known are the so-called intrinsically photosensitive retinal ganglion cells (ipRGCs) that express a visual pigment, melanopsin, with closer homology to invertebrate opsins than to rod or cone opsins (Provencio et al. 2000). Along with a variety of other molecular similarities, this suggests that ipRGCs share a common evolutionary origin with the microvillar photoreceptors of Drosophila and most invertebrates (Arendt 2003).

Unlike rods and cones, which hyperpolarise in response to illumination, ipRGCs depolarise slowly, generating action potentials that adapt at best sluggishly thus giving an output that reflects the intensity of ambient illumination (Berson 2003; Do and Yau 2010; Fu et al. 2005; Hankins et al. 2008). This seems suited to their major functions of controlling the pupillary light reflex and entraining circadian rhythms via projections to the olivary pretectal nucleus and suprachiasmatic nucleus. Until recently there were only hints as to the underlying transduction cascade. For example, when expressed in Xenopus oocytes, melanopsin is capable of activating a Gq-based PLC cascade and a mammalian TRPC channel (Panda et al. 2005). In native ipRGCs the light-activated current can be blocked by PLC inhibitors (Graham et al. 2008) and inhibitors (albeit rather unspecific) of TRPC channels (Warren et al. 2006). Most convincingly however, it was recently demonstrated that the light-activated current in native ipRGCs was abolished in mouse knockouts lacking PLCβ4 as well as double knockouts lacking both TRPC6 and TRPC7. Interestingly, PLCβ4 is more closely related to PLC in Drosophila photoreceptors (norpA) than it is to other vertebrate PLC isoforms (Ferreira and Pak 1994). Along with the apparent involvement of TRPC channels, there are thus close parallels with phototransduction in Drosophila. There is, as yet, little or no evidence for the downstream pathway linking PLC activation to TRPC channel activation in the ipRGCs. In heterologous expression systems TRPC6 and TRPC7 can be activated by DAG (Hofmann et al. 1999; Okada et al. 1999). Putative native TRPC6/7 channels, in other mammalian cells, such as the vascular smooth muscle, are also reported to be activated by DAG, possibly in synergy with InsP3 and PIP2 depletion (Albert and Large 2003, 2006; Ju et al. 2010).

9.2 TRPA1

Finally, recent studies in both vertebrate and invertebrates have suggested roles for TRPA1 channels in some atypical “dermal photoreceptors”.

9.2.1 Melanocytes

Melanin synthesis in human epidermal melanocytes (HEM) is stimulated by absorption of UV light. Short-wavelength (UVB) light may stimulate melanin synthesis downstream of DNA damage itself (Lin and Fisher 2007), but the mechanism underlying response to longer-wavelength UVA light is poorly understood. In a recent study UVA light was shown to activate a current in HEM cells, which was inhibited by TRPA1 antagonists (camphor or HC-030031) and almost eliminated by TRPA1 miRNA (Bellono et al. 2013). Both miRNA and TRPA1 antagonists also inhibited early melanin synthesis. Based on pharmacology, activation appeared to be via a G protein and PLC-based signalling pathway. It also required pre-incubation of cells with retinaldehyde suggesting the involvement of an opsin-based pigment (Bellono and Oancea 2013; Wicks et al. 2011).

9.2.2 Larval Photo-Avoidance Behaviour

Drosophila larvae have a primitive eye (Bolwig’s organ), which mediates larval phototaxis. However, even after these have been genetically deleted, larvae were shown to avoid bright UV light. This photo-avoidance behaviour is mediated by a class of sensory neuron (class IV neurons) with dendrites that tile essentially the entire body surface and was abolished in mutants lacking TRPA1 channels (Xiang et al. 2010). The response also requires an orphan GPCR (Gr28b), which is annotated as a gustatory receptor. Whether this represents a previously unrecognised visual pigment or whether it might respond to a chemical generated via UV illumination is unclear, as is the transduction pathway linking the receptor to TRPA1 activation. Interestingly, a Caenorhabditis elegans homologue of Gr28b—lite1—was also found to be required for UV photo-avoidance behaviour in C. elegans; however, in this case the transduction cascade involves guanylate cyclase culminating in activation of cGMP-gated channels (Liu et al. 2010).

Concluding Remarks

The unique experimental advantages of the Drosophila retina for both genetic and functional analysis not only led to the discovery of the TRP ion channel family but have also provided key insight into many aspects of TRP-channel function. From its discovery it was clear that Drosophila TRP was activated downstream of PLC (Hardie and Minke 1992). This is now regarded as a common feature for all TRPC channels, and in fact many, if not most, other TRP channels can be regulated in one way or another by PLC. Nevertheless, exactly which consequence of PLC activity is responsible for activation is often far from clear. TRPC2, 3, 6 and 7 are reportedly activated by DAG (Hofmann et al. 1999; Lucas et al. 2003; Okada et al. 1999); but whether this action is direct remains debated (Lemonnier et al. 2008), and no DAG binding domain has been identified. Roles for InsP3 and PIP2 have also been reported (Ju et al. 2010). The mechanism of activation of TRPC4, 5 and 1 is yet more mysterious, though complex roles for PIP2 have been proposed (Otsuguro et al. 2008; Trebak et al. 2009). Even though it is debatable whether PUFAs are physiologically relevant agonists in the photoreceptors, the discovery that TRP and TRPL could be activated by PUFAs was the first indication that TRPC channels were regulated by lipid messengers (Chyb et al. 1999; Hardie 2003). This is again believed to be a common feature of not only all TRPCs but also many other TRPs (Beech 2012; Hardie 2007).

Whether the latest working hypothesis for physiological activation of TRP and TRPL, a combination of membrane tension and protons (Hardie and Franze 2012), is more generally applicable remains to be seen. However, mechanical gating has been repeatedly suggested, albeit often controversially, for many members of the TRP family (Barritt and Rychkov 2005; Christensen and Corey 2007; Liedtke 2007; Lin and Corey 2005; Maroto et al. 2005; Mederos y Schnitzler et al. 2008; Patel et al. 2010; Quick et al. 2012; Sharif-Naeini et al. 2008; Spassova et al. 2006; Su et al. 2007; Yin and Kuebler 2010). Protons are also known modulators of several TRP channels, and although most reports concern extracellular sites—as in TRPV1 and TRPC5 (Ryu et al. 2007; Semtner et al. 2007)—intracellular protons have been implicated as inhibitors of gating of TRPM2 (Du et al. 2009) and activation of TRPA1 (Wang et al. 2010). More generally, like many, if not all, TRP channels, Drosophila TRP and TRPL are polymodally regulated, with multiple signals including lipids, protons, Ca2+ and membrane tension all playing potentially important roles.

Although the physiological roles of many vertebrate TRP channels are still often only poorly understood, along with the electrical signal (depolarisation), Ca2+ influx is probably the single most important physiological consequence of TRP-channel activation. Again, for the light-sensitive Drosophila TRP channel, this has been clear from its discovery, with Ca2+ influx having profound roles acting on multiple targets, with actions ranging from the millisecond timescale of the light response to long-term cell survival or degeneration. Nevertheless, as with all members of the TRP family, many questions remain to be answered, not least, a final resolution to the question of how the channels are activated. The mechanism of dual (positive and negative) Ca2+-dependent regulation is also surprisingly poorly understood, as is the role of extensive phosphorylation of both proteins.