Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP3. trp is shown to encode a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the Trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells (Montell, 1989).
Invertebrate phototransduction is an important model system for studying the ubiquitous inositol-lipid signaling system. In the transient receptor potential (trp) mutant, one of the most intensively studied transduction mutants of Drosophila, the light response quickly declines to baseline during prolonged intense light. Using whole-cell recordings from Drosophila photoreceptors, the wild-type response is shown to be mediated by at least two functionally distinct classes of light-sensitive channels and both the trp mutation and a Ca2+ channel blocker (La3+) selectively abolish one class of channel with high Ca2+ permeability. Evidence is also presented that Ca2+ is necessary for excitation and that Ca2+ depletion mimics the trp phenotype. It is concluded that the recently sequenced Trp protein represents a class of light-sensitive channels required for inositide-mediated Ca2+ entry, and it is suggested that this process is necessary for maintained excitation during intense illumination in fly photoreceptors (Hardie, 1992).
Phototransduction in Drosophila is mediated by the ubiquitous phosphoinositide cascade, leading to opening of the TRP and TRPL channels, which are prototypical members of a novel class of membrane proteins. Drosophila mutants lacking the TRP protein display a response to light that declines to the dark level during illumination. It has recently been suggested that this response inactivation results from a negative feedback by calcium-calmodulin, leading to closure of the TRPL channels. It is also suggested that in contrast to other phosphoinositide-mediated systems, Ca2+ release from internal stores is neither involved in channel activation nor in phototransduction in general. This study shows that inactivation of the light response in Trp photoreceptors is enhanced upon reduction of the intracellular Ca2+ concentration. Furthermore, in Ca(2+)-free medium, when there is no Ca2+ influx into the photoreceptors, a significant elevation of intracellular Ca2+ is seen upon illumination. This elevation correlates with ability of the cells to respond to light. Accordingly, malfunctioning of Ca2+ stores, either by Ca2+ deprivation or by application of the Ca2+ pump inhibitor, thapsigargin, confers a trp phenotype on wild type flies. The results indicate that the response inactivation in trp cells results from Ca2+ deficiency rather than from Ca(2+)-dependent negative feedback. The results also indicate that there is light-induced release of Ca2+ from intracellular stores. Furthermore, the response to light is correlated to Ca2+ release, and normal function of the stores is required for prolonged excitation. It is suggested that phototransduction in Drosophila depends on Ca(2+)-release mediated signalling and that TRP is essential for the normal function of this process (Cook, 1999).
The Drosophila trp gene encodes a light-activated Ca(2+) channel subunit that is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. A new mutant, Trp(P365), is described that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. In spite of its unusual phenotypes, Trp(P365) is a trp allele because a Trp(P365) transgene induces the mutant phenotype in a wild-type background, and a wild-type trp transgene in a Trp(P365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp(P365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp(P365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain (Yoon, 2000).
The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. A single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel. The following are some tentative conclusions from this study: (1) Trp(P365) is important for the regulation of TRP channel opening; (2) The particular amino acid residue preferred for this position appears to be different for different species (Phe for Drosophila and Leu for mammals) perhaps because of slightly different channel environments; (3) Ile appears to be particularly poorly tolerated at this position. It is concluded that the residues at positions 550 or in its immediate vicinity may be in a position to critically affect channel gating (Hong, 2002).
To identify new alleles of the trp locus, a screen was performed for the recently isolated collection of chemically induced third chromosome mutations, which display defects in the electroretinogram (ERG) recording (for details see T. Wang, 2005a). Exposure of wild-type flies to light results in two discriminable components in the ERG. These include a sustained corneal negative maintained component arising from responses of all retinal cells (photoreceptor cells and pigment cells) and on- and off-transients emanating from activity in the second-order neurons in the optic lobes. The classic trp phenotype is characterized by a transient response to light, resulting from rapid light-dependent inactivation of the remaining Trpl cation channel (T. Wang, 2005b).
Each of the third chromosome mutations was crossed to the strong trpP343 allele and five were identified that failed to complement the recessive Trp phenotype: therefore, they represented new trp alleles. Four of the new trp alleles exhibited a transient ERG phenotype indistinguishable from trpP343, whereas the phenotype of the fifth (trp14) was distinct in that the decline in the light response was much slower than in trpP343 or other alleles isolated in this or previous studies. The trp14 phenotype was due to an autonomous defect in the photoreceptor cells, rather than the pigment cells, since the slower decline in the receptor potential was evident in single photoreceptor cells assayed by performing intracellular recordings (T. Wang, 2005b).
Currently, all of the existing loss-of-function mutations cause large reductions in protein levels, although the molecular lesions have not been defined. Among the extant trp alleles, the one with the strongest phenotype is trpP343. The trpP343 genomic region was sequenced and it was found that the Trp protein coding region was identical to wild type. Rather, there was a mutation in a conserved 5' splice site that was essential for mRNA splicing. The mutation presumably results in instability of the mRNA, since no trpP343 mRNA is detected (Montell, 1989). Given the strong phenotype and lack of mRNA or Trp protein expressed in trpP343 (Montell, 1989), this allele would appear to represent a null (T. Wang, 2005b).
To examine the levels of Trp protein expressed in the new alleles described in this study, Western blots were performed. Among those alleles that displayed a phenotype typical of trpP343, three did not express any detectable Trp protein (trp38, trp74, and trp92), whereas a fourth expressed very low levels of Trp (trp47). This was in contrast to trp14 flies in which Trp was expressed at ~60% the level as in wild type. Other rhabdomeral proteins including INAD, INAC, NORPA, and Rh1 (rhodopsin 1) were expressed at comparable levels in trp14, trpP343, and wild-type flies (T. Wang, 2005b).
To determine the molecular defects associated with the five new trp alleles, the genomic DNA was sequenced and the sequences were compared to that of the original isogenized stock used to conduct the mutagenesis. Among the four alleles that expressed very low or no detectable Trp, one had a frameshift mutation resulting in premature translation termination (trp92), two had missense mutations (trp47 and trp74), and one had no mutation in the transcribed region and therefore may contain a mutation affecting the trp promoter. The allele (trp14) expressing Trp at 60% wild-type levels, and which exhibited a phenotype distinct from other trp alleles, had two missense mutations. One of these mutations changed an amino acid in the pore loop between transmembrane domains five and six (residue 612; leucine to phenylalanine), whereas the other was situated between the sixth transmembrane segment and a highly conserved sequence referred to as Trp box 1 (residue 671; arginine to glutamine) (T. Wang, 2005b).
Given that Trp has dual functions as a molecular anchor and as a cation channel (Li, 2000; Tsunoda, 2001), whether the Trp phenotype in trp14 was a consequence of perturbation of the anchoring role was considered. Drosophila compound eyes consist of ~800 repetitive units, referred to as ommatidia, each of which includes six outer photoreceptor cells (R1-6) and a central R7 or R8 cell in the distal region of the retina. Each photoreceptor cell contains a microvillar segment, the rhabdomere, where most of the proteins that function in phototransduction, such as the core members of the signalplex, are concentrated. These include Trp, protein kinase C (INAC), phospholipase C (NORPA), and INAD. Mutations that eliminate or disrupt the anchoring role of Trp result in mislocalization of these core members such that they are present in both the rhabdomeres and cell bodies (Chevesich, 1997; Tsunoda, 1997; Li, 2000; Tsunoda, 2001). However, other INAD binding partners, such as Rh1, do not depend on Trp for normal localization (Li, 2000). In trp14 photoreceptor cells, each of the core and other rhabdomeral proteins examined displayed a rhabdomere localization pattern indistinguishable from wild type. Consistent with these data, it was found that INAD coimmunoprecipitated effectively with the Trp14 protein. These data indicate that the Trp phenotype in trp14 flies is not due to an alteration in the Trp anchoring function. Rather, they raise the possibility that the phenotype is due to a defect in Trp channel function (T. Wang, 2005b).
In trp flies such as trpP343 the transient potential ERG phenotype is a consequence of inactivation of the Trpl channel during constant light stimulation. However, in Trpl mutant flies, which express Trp but not Trpl, the ERG response is maintained during a typical 5-10-s light pulse. To examine Trp14 channel function independent of Trpl, the trp14 allele was introduced into a Trpl-null mutant (Trpl302) background. As previously shown, Trpl302;trpP343 flies are blind, since they do not express Trpl or Trp (Niemeyer, 1996; Reuss, 1997). Flies harboring just the Trpl302 mutation show a response to 10 s of light similar to wild type, because these flies express wild-type Trp. In contrast, Trpl302;trp14 flies displayed a Trp phenotype similar to trp14. These data indicate that the transient light response in trp14 flies is due to disruption of Trp channel function (T. Wang, 2005b).
To exclude the possibility that the trp14 phenotype is a consequence of mislocalization of Trp14, the mutant protein was spatially localized. Immunostaining experiments were performed and it was found that Trp14 was detected exclusively in the rhabdomeres, as is the case for wild-type Trp. Therefore, the transient light response in trp14 is not due to mislocalization of the Trp protein (T. Wang, 2005b).
Given that trp14 flies express a 40% lower concentration of Trp, whether a transient light response could be induced by expression of low levels of Trp was tested. Therefore, transgenic flies that expressed varying levels of wild-type Trp under the control of the heat-shock promoter (hs-trp) were generated and the transgene was placed in a Trpl302;trpP343 genetic background. Even though use of the heat shock protein 70 (hsp70) promoter typically results in widespread expression, the Trp protein expressed under the control of the hsp70 promoter was found exclusively in the retina and not in the optic lobes or elsewhere in the adult head. To induce different low levels of Trp, the hs-trp flies were exposed to 30, 60, and 120 min heat-shock treatments, which resulted in the production of ~4%, 7%, and 10% of the wild-type levels of Trp, respectively. Expression of only ~10% the normal concentration of Trp restored an ERG in Trpl302;trpP343 flies, which was not transient. Even 4% the normal levels of Trp did not cause a transient light response similar to trp14, although the amplitude of the ERG was reduced. These data indicate that the trp14 mutant phenotype was due to the mutation of the Trp14 protein, rather than simply due to expression of low levels or mislocalized Trp protein (T. Wang, 2005b).
Strong loss-of-function mutations in trp, such as in trpP343, result in light dependent retinal degeneration. Considering Trp has dual channel and nonchannel roles, disruption of either function could potentially cause retinal degeneration. To address which of these two roles is more critical to prevent retinal cell death, the morphology was examined of trpDelta1272 (Li, 2000: and trp14 ommatidia, which display specific defects in the scaffold and Trp channel functions, respectively. Wild-type ommatidia contain eight photoreceptor cell rhabdomeres, seven of which are present in any given plane regardless of their age or whether the flies were maintained in the dark or under a light-dark cycle (T. Wang, 2005b).
The retinal degeneration in trp14 and trpP343 was much more severe than that in trpDelta1272 flies. In both trpP343 and trp14 flies, the rhabdomeres began to disappear between 7 and 10 d after eclosion and almost no rhabdomeres remained after 14 d of exposure to a 12-h light-12-h dark cycle. By contrast most trpDelta1272 flies maintain a full complement of seven rhabdomeres after 14 d of a light-dark cycle. Nevertheless, the size of the rhabdomeres was typically smaller than in similarly aged wild-type and large intracellular vacuoles were present inside the cell bodies indicating that retinal degeneration had initiated. By 30 d after eclosion, most of the rhabdomeres in these flies had degenerated. Thus, the retinal degeneration is much more severe in trpP343 and trp14 than trpDelta1272, indicating that the Trp channel function rather than the scaffold function is more critical to prevent the retinal cell death (T. Wang, 2005b).
The retinal degeneration resulting from defects in Trp function was suppressed by maintaining the flies in the dark; this keeps the Trp channels in an inactive state. To assess the extent of suppression, the flies were maintained in the dark for 30 d, which was more than twice as long the 14-d light-dark cycle that caused elimination of almost all rhabdomeres in trp14 or trpP343 flies. In dark-maintained trpDelta1272 flies, seven rhabdomeres were present in ommatidia, although the size of the rhabdomeres was reduced. Indistinguishable results were obtained with trpP343. The suppression of retinal degeneration was even more complete with trp14, as all ommatidia contained a full set of seven rhabdomeres of normal size. This result was striking since the retinal degeneration occurring under a light-dark cycle is significantly more rapid in trp14 than in trpDelta1272 (T. Wang, 2005b).
To explore the mechanism underlying the retinal degeneration in trp mutants, a genetic approach was used. The combination of results described above indicates that a defect in Trp channel function underlies the retinal degeneration in trp flies. If the basis of the retinal degeneration in the trp mutant was due to diminished Ca2+ influx during light stimulation, then the cell death might be reduced by mutations in the Na+/Ca2+ exchanger, CalX, which functions in Ca2+ extrusion in photoreceptor cells (T. Wang, 2005b).
To test whether calx can suppress the retinal degeneration in trpP343 and trp14, the morphology of calxB,trpP343, and calxB,trp14 compound eyes was examined. trp14 or trpP343 flies maintained under a light-dark cycle for 14 d displayed nearly complete loss of the rhabdomeres. The retinal degeneration in calxB flies was even more severe; there were few rhabdomeres left after a 7 d light-dark cycle and almost no rhabdomeres left after a 14-d light-dark cycle. In contrast, most ommatidia from either calxB,trpP343 or calxB,trp14 double mutant flies contained all the rhabdomeres after 14 d under a light-dark cycle. Moreover, the core signalplex proteins, NORPA, INAC, and INAD were mislocalized in calxB,trpP343 flies, indicating that introducing the calx mutation does not prevent loss of the Trp scaffold function in trpP343. The effect of calx on trp was specific because the calxB mutation did not suppress the cell death resulting from mutations in other phototransduction genes such as inaC, which encodes an eye-enriched PKC. These results indicated that the retinal degeneration in trp14 or trpP343 is a consequence of decreased intracellular Ca2+ levels, whereas the photoreceptor cell death in the calx mutant results from Ca2+ overload (T. Wang, 2005b).
To explore further the mechanism of the retinal degeneration in trp14 and trpP343, tests were made to see whether it could be suppressed by mutations in the gene encoding the major arrestin (arrestin2 -- arr2). Elimination of Arr2 reduces the retinal degeneration associated with certain mutations, such as norpA (disrupts phospholipase C), which prevents light-dependent activation of Trp channels. The retinal degeneration in norpA results from formation of stable rhodopsin-arrestin complexes and subsequent endocytosis of rhodopsin (T. Wang, 2005b). It was found that strong mutations in arr2 partially suppress the retinal degeneration in trp14 and trpP343 flies. Whereas a 14-d exposure to a light-dark cycle results in extensive loss of rhabdomeres in arr25,trpP343 or trp14 eyes, most ommatidia in arr25,trpP343 or arr25,trp14 double mutants contained seven rhabdomeres. However, the sizes of the rhabdomeres were significantly reduced (T. Wang, 2005b).
The two mutations in trp14 alter residues in the pore loop or immediately NH2 terminal to Trp box 1 (residues 612 and 671, respectively). Given the potential effects of a pore-loop mutation on ion selectivity and the highly conserved nature of the region between the sixth transmembrane segment (TM6) and the Trp domain (Montell, 2005b), both of these mutations were in intriguing positions that could potentially be responsible for the trp14 phenotype (T. Wang, 2005b).
To determine whether one or both mutations were responsible for the transient light response phenotype in trp14, transgenic flies expressing Trp isoforms with just the L612F or R671Q amino acid substitution (trp612F and trp671Q, respectively) were generated and tested. The wild-type or mutant trp cDNAs were fused to the ninaE (neither inactivation nor afterpotential E [encodes Rh1]) promoter and introduced into the Trpl302;trpP343 double mutant background. Subsequently Western blots were performed on the transgenic flies demonstrating that wild-type Trp, Trp612F, and Trp671Q were all expressed at similar levels (T. Wang, 2005b).
It was found that the missense mutation juxtaposed to the Trp domain was responsible for the phenotype in trp14. The Trpl;ninaE-trp612F,trpP343 flies displayed a wild-type ERG response indistinguishable from Trpl;ninaE-trpwt,trpP343. Conversely, the Trpl;ninaE-trp671Q,trpP343 flies showed a transient ERG phenotype. Moreover, the trp671Q flies exhibited an ERG phenotype with a more rapid decline typical of trpP343, suggesting that the 612F mutation resulted in a slight suppression of the Trp phenotype. These data demonstrate that the missense mutation at residue 671 situated between the sixth transmembrane domain and Trp box 1 (Montell, 2005b) was the key mutation responsible for the trp14 phenotype (T. Wang, 2005b).
The residues in Trpl and Trpgamma, corresponding to the required arginine 671 in Trp, are also basic amino acids (histidine 678 and arginine 662, respectively) suggesting that a basic residue at this position flanking the Trp domain is essential in the Drosophila TrpC channels. Therefore, transgenic flies were generated expressing derivatives of Trpl in which histidine 678 was replaced either with a conservative arginine substitution (Trpl678R) or with an uncharged glutamine (Trpl678Q). The mutant and wild-type Trpl cDNAs were fused to the ninaE promoter and introduced into a Trpl302;trpP343 background. The transgenic Trpl proteins were all expressed at similar levels, though at an approximate sevenfold higher level than in wild-type due to the strong ninaE promoter (T. Wang, 2005b).
To determine the consequences of the mutations in Trpl, ERGs were performed after introducing the transgenes in a Trpl302;trpP343 background. Whereas the double Trpl302;trpP343 mutant was blind, overexpression of the wild-type Trpl in this genetic background (Trplwt) restored a transient response to light indistinguishable from trpP343. Furthermore, introduction of the Trpl678R transgene into the genome of Trpl302;trpP343 flies resulted in an ERG response similar to Trplwt. Thus, replacing histidine 678 with an arginine did not disrupt Trpl function. However, the amplitude of the ERG was significantly reduced in Trpl302;trpP343 flies expressing the Trpl678Q transgene with the histidine to glutamine substitution in residue 678. The combination of these results demonstrates a critical role of a basic residue at the corresponding positions in Trp and Trpl, flanking the highly conserved Trp box 1 (T. Wang, 2005b).
Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. The TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. This study reports that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, allowing TRPL-eGFP translocation to be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Gαq, phospholipase Cß and the TRP ion channel, or in proteins required for TRP function. These data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, it was found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca2+-influx in the translocation process. The critical role of Ca2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca2+ (Meyer, 2006; full text of article).
Besides TRPL, at least two other proteins mediating Drosophila phototransduction, Arrestin 2 and the visual Gαq, undergo light-dependent translocation between the rhabdomere and the cell body. Likewise, in vertebrate photoreceptors arrestin and the visual G protein transducin translocate between the inner and outer segment in a light-dependent way. In both visual systems, arrestin and G protein movements occur in opposite directions, that is, in the light arrestin accumulates whereas the G protein is depleted in the photoreceptive membrane and vice versa in the dark. Accordingly, these light-dependent relocations of visual signaling proteins make the photoreceptor more sensitive in the dark and less sensitive in the light and mediate long-term adaptation of the Drosophila and vertebrate visual systems. A third protein that translocates in vertebrate photoreceptors is the Ca2+ binding protein recoverin (Meyer, 2006).
The mechanisms underlying these protein translocations have been elucidated in part for Drosophila arrestin, the Gαq subunit and for vertebrate transducin. Translocation of Drosophila arrestin from the cell body to the rhabdomere has been reported to require the ninaC-encoded myosin III which may actively transport arrestin along the actin cytoskeleton of the photoreceptor microvilli through PIP3-enriched vesicles, to which arrestin binds. However, the requirement of the myosin III NINAC for arrestin translocation has been challenged in a more recent publication. Removal of arrestin from the rhabdomeral membranes in the dark does not require cytoskeletal elements and may thus occur passively. Likewise, Gαq translocation into the rhabdomere, but not its removal, is facilitated by the myosin III NINAC. Translocation of vertebrate transducin is aided by phosducin, an abundant photoreceptor-specific protein that binds to the ßγ subunits of transducin. Phosducin increases the solubility of the G protein subunits and may thereby facilitate transducin translocation (Meyer, 2006).
These mechanisms are markedly different from the mechanism underlying TRPL translocation because TRPL is a transmembrane protein that cannot enter the soluble fraction and needs to be removed from the rhabdomere by an endocytotic pathway, whereas arrestin and the visual G protein change from a membrane attached state to a soluble state. Therefore, elucidating the triggering mechanism of TRPL translocation reported in the present study is the first step for unraveling the mechanism underlying an important cellular process (Meyer, 2006).
In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses (Leung, 2008).
Visual transduction in Drosophila utilizes a G protein-coupled, phospholipase C-mediated signaling cascade. Phospholipase C, upon activation via rhodopsin and G protein, Gq, catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into two potential second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP3). A body of evidence suggests that IP3 is not involved in Drosophila phototransduction, leaving the DAG branch as a likely source of messenger(s) of activation for the phototransduction channels, transient receptor potential (TRP) and TRP-like (TRPL). The mechanism by which the diacylglycerol (DAG) branch might activate the TRP/TRPL channels is still unresolved. The first indication that a lipid messenger might be involved was provided by Chyb (1999), who showed that polyunsaturated fatty acids (PUFAs) could activate both TRP and TRPL channels either in intact photoreceptors or heterologous expression systems. Later, evidence was provided that DAG is required for photoreceptor excitation using DAG kinase mutants, rdgA. Because the conversion of DAG to phosphatidic acid is blocked in these mutants, they should have an elevated DAG basal level. TRP/TRPL channels are constitutively active in rdgA, and diminished responses of hypomorphic PLC (norpA) mutants can be greatly enhanced by rdgA mutations, in support of the contention that DAG might be excitatory to the channels. However, rdgA mutations are expected to raise the basal levels of not only DAG but also its metabolites. In addition to these two molecules, phosphatidylinositol 4,5-bisphosphate (PIP2) has also been suggested to play a role in channel excitation. Currently, no consensus exists as to which, if any, of these might be the excitatory agent for TRP/TRPL channels (Leung, 2008).
Drosophila TRP is the founding member of a superfamily of TRP channel proteins. There are now nearly 30 mammalian members of this superfamily comprising seven subfamilies. Although these channels are heterogeneous in their modes of activation, at least four mammalian TRP channels have been reported to be activated by DAG: TRPC2, -3, -6, and -7. While there may be variations in the mechanisms of activation of these channels, elucidation of Drosophila TRP/TRPL channel activation could provide insight into activation of these channels as well (Leung, 2008).
Because both DAG and its potential metabolite, PUFA, have been implicated in the activation of TRP/TRPL channels, a key enzyme in this process is likely to be DAG lipase, which catalyzes the hydrolysis of DAG. Little is known about DAG lipases. Two mammalian DAG lipase genes, DAGLα and -β, have been identified by a bioinformatics approach and characterized both biochemically and molecularly), and many proteins homologous to DAGα and -β have been identified across species. In the case of Drosophila, , rolling blackout (rbo) has been suggested to be in a DAG lipase gene. The protein encoded by the rbo gene, however, shows little homology to the known mammalian DAG lipases. Moreover, conditional loss of the RBO protein leads to rapid depletion of DAG, the opposite of what one would expect if RBO catalyzes the hydrolysis of DAG. Furthermore, in the absence of previous activity, the receptor potential is normal in rbo mutants, making it unlikely that RBO has any direct involvement in the activation of TRP/TRPL channels. Other than rbo, no candidate DAG lipase that might function in phototransduction has been reported in any species (Leung, 2008).
This work reports on a Drosophila DAG lipase (DAGL) gene, inaE, identified from mutants that are defective in photoreceptor responses to light. The protein isoforms encoded by this gene show high sequence similarity to the two known mammalian DAGLs, exhibit DAGL activity in vitro, are highly expressed in photoreceptors, and have access to rhabdomeres. Genetic evidence suggests that the inaE-encoded DAGLs interact in vivo with the DAG generated in the phototransduction cascade. Analysis of mutants generated by imprecise excision of P element insertion in inaE show that no physiologically meaningful photoreceptor responses can be generated if inaE gene is severely impaired (Leung, 2008).
The inaE gene was identified by two ethylmethane sulfonate (EMS)-induced allelic mutants: N125 and P19. These mutants are characterized by their 'ina' (inactivation, no afterpotential) electroretinogram (ERG) phenotype. Wild-type flies, when placed on a white-eye (w) background, respond to a bright blue stimulus with a large response during light stimulus followed by a prolonged depolarizing afterpotential (PDA) after the light is turned off. A second blue stimulus elicits only a small response, originating from R7/8 photoreceptors, superposed on the PDA. By contrast, in ina mutants, the response begins to decay during stimulus (inactivation), and the decay continues after the stimulus. As a result, the PDA is greatly diminished in amplitude (no afterpotential). This phenotype can also be viewed as a mild form of the 'trp' phenotype displayed by strong mutants of the TRP channel gene. In trp mutants, the response to the first blue stimulus decays nearly to baseline during stimulus, and there is no PDA (Leung, 2008).
Moreover, responses of inaE mutants resemble those of trp in that they both display refractory properties. Following a response to the first stimulus, only very small responses can be elicited from trp until they recover over a period of minutes, while wild-type responses recover almost immediately. Likewise, inaE mutants exhibit a similar refractory period, although the degree and duration of response suppression are not as pronounced or prolonged as in trp (Leung, 2008).
In addition to the above similarities, inaEN125 acts as a genetic enhancer of TrpP365. TrpP365 is a semidominant allele of trp, which causes constitutive activation of TRP channels and, as a result, massive photoreceptor degeneration from excessive Ca2+ influx. In TrpP365 homozygotes, degeneration is already so advanced in 1- to 2-day-old flies that essentially no ERGs can be elicited. TrpP365 heterozygotes exhibit a much milder phenotype and elicit ERGs of substantial amplitude at the same age. However, if inaEN125 is introduced into the TrpP365/+ background, the resulting phenotype is as severe as that of TrpP365 homozygotes. The genetic enhancement of TrpP365/+ by inaEN125 and the basic similarity of ERG phenotypes between inaE and trp mutants led to the hypothesis that the protein products of these two genes may interact and/or subserve closely related functions (Leung, 2008).
The CG33174 gene had not been characterized previously, and its function was electronically inferred as 'triacylglycerol lipase (TAGL) activity'. However, the possibility is considered that the above annotation could simply reflect a dearth of information on DAGLs. The first two human DAGLs, DAGLα and -β, were cloned and characterized by a bioinformatic approach and shown to be sn-1 type DAGLs. Multiple alignment of INAE-A and INAE-D with DAGLα and -β revealed extensive sequence and domain conservations. All four proteins are predicted to have four transmembrane segments near the N-terminal region, and they all have a lipase_3 domain with a highly conserved serine active site. Overall sequence homology between INAE-D and the two human DAGLs is 39% identity and 56% similarity for DAGLα and 30% identity and 50% similarity for DAGLβ, respectively. In the lipase_3 domain, the sequence homology between INAE-A/D and the mammalian proteins rises to 60% and 45% identity and 73% and 63% similarity for DAGLα and DAGLβ, respectively (Leung, 2008).
To demonstrate that the INAE proteins have DAG lipase activity, the INAE-A and -D protein isoforms were expressed in E. coli and purified to >95% purity to carry out DAG lipase assays using 1-stearoyl-2-arachidonoyl-sn-glycerol as substrate, and the lipase assay products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) for identification of hydrolysis products and kinetic studies (Leung, 2008).
LC-MS detected four products from the analysis of both INAE isoforms: two primary products, stearic acid (18:0) and 2-arachidonoyl glycerol (2-AG), and two minor products, arachidonic acid (20:4) and 1-stearoyl glycerol (1-SG), eluting at 6.3, 9.3, 5.8, and 8.7 min, respectively. Two primary products corresponded to hydrolysis at the sn-1 position of DAG substrate, and the two minor products corresponded to hydrolysis at the sn-2 position. Thus, in vitro, the two recombinant INAE isoforms are both DAG lipases highly preferential for hydrolysis at the sn-1 position, with the D form having much higher activity than the A form (Leung, 2008).
A growing body of evidence suggests that Drosophila phototransduction utilizes the DAG branch of the G protein-coupled, PLCβ-mediated signaling pathway). Although DAG lipase is expected to play a critical role in this pathway, no DAG lipase that could play such a role had been identified previously in Drosophila. This study reports on a DAG lipase identified from the Drosophila mutants, inaE. The inaE gene was found to encode two protein isoforms, INAE-A and INAE-D, by alternative splicing. Both of these proteins are highly homologous to the two previously identified mammalian sn-1 type DAG lipases, and in vitro DAG lipase assays of recombinant INAE-A and INAE-D showed that both are DAG lipases highly preferential for hydrolysis at the sn-1 position. Expression of the INAE protein is not restricted to the eye but occurs throughout the head, consistent with the finding that strong mutations in this gene are homozygous lethal. In photoreceptors, anti-INAE antibody labeling occurs as punctate staining scattered throughout the photoreceptor cytoplasm. Occasionally, some of the puncta are found within the rhabdomeres, indicating that some DAGL enters the rhabdomeres. Results of the norpA inaE double mutant study provide strong functional support for the above observation. In this study, the receptor potential disappears in an inaE allele-dependent manner -- the stronger the inaE allele in the double mutant, the more severe the double mutant phenotype. The allele dependence strongly suggests that the action of inaE-encoded DAGL is responsible for the observed double mutant phenotype. Furthermore, to affect the receptor potential phenotype, DAGL must act on the DAG generated by norpA-encoded PLCβ, and, for that to occur, DAGL must enter the rhabdomeres (Leung, 2008).
Because inaE mutations already available were all relatively mild, severe mutations were generated by imprecise excisions of a P element insertion in the inaE gene. These imprecise excision alleles were homozygous lethal and had to be studied as eye mosaics. Quantitative RT-PCR results showed that even the severest of these imprecise excision mutants, inaExl18, is not a null mutant and expresses RNA at ~25% of the normal level. This mutation profoundly affects the photoreceptor responses to light. If xl18 is placed on a norpAH43 background to reduce the amount of DAG generated, the light stimulus generates no response at all. In xl18 flies themselves, a bright prolonged stimulus generates only a small response of slow kinetics that decays to baseline completely during the stimulus. This response most likely represents the residual DAGL activity in this severely affected mutant. As the severity of mutation progressively decreases in the xl series of mutants, the receptor potential phenotype returns to normal in an allele-dependent manner. Again, the inaE allele dependence strongly argues that the action of inaE-encoded DAGL is responsible for the observed change in the receptor potential phenotype. These results, taken together, suggest (1) that the production of DAG metabolite(s) through the action of the inaE-encoded DAGL is required for the generation of photoreceptor responses to light and (2) that, in the absence of the metabolite, DAG plays little direct role in the activation of channels. However, the identity of the excitatory molecule cannot be specified from this work. It could be one or more of the products generated by INAE, such as monoacylglycerol (2-AG) or stearic acid or even DAGL (INAE) itself (Leung, 2008).
While DAG may not have a direct role in channel activation, evidence was found suggesting that it may be important in regulating the action of the DAG metabolite that acts as an excitatory agent, although the evidence is still largely indirect. The ability of inaEN125 to act as an enhancer of TrpP365/+ seems to present a quandary when considered in relation to the results summarized above. If DAG has little or no direct role in channel activation, how does one explain the disappearance of the small response present in P365/+ when N125 is added to this background? A simple explanation for the phenomenon would be that DAG is excitatory to the channels and that adding N125 to the P365/+ background raised the level of DAG to make more channels to become constitutively active in N125;P365/+ than in P365/+. However, results of the experiment replacing N125 with a stronger inaE allele, xl18, in the N125;P365/+ double mutant run counter to this simple explanation. Replacing N125 with xl18 should have sharply raised the basal DAG level further in the double mutant. If DAG were excitatory, the resting potential should have depolarized even more than before the N125 replacement, and no receptor potential at all should have been obtained. Just the opposite results were obtained. A small but distinct receptor potential could be recorded from xl18;P365/+, and the resting potential has returned to the level in P365/+. These results are incompatible with the hypothesis that DAG is excitatory to the channel and instead provide another line of evidence for the conclusions summarized earlier (Leung, 2008).
However, the fact that a much more severe phenotype is obtained in N125;P365/+ than in P365/+ or xl18;P365/+ suggests that DAG may have a role in facilitating, enhancing, and orchestrating the action of the DAG metabolite that serves as the excitatory agent. This action of DAG would be more noticeable under conditions in which a sufficient amount of the excitatory product is produced, as in N125;P365/+ rather than in xl18;P365/+. A similar action of DAG can also be inferred from the norpA inaE double mutant studies. In this series of experiments, hypomorphic norpA mutation, H43, was used to restrict the amount of DAG generated both in the single and double mutants. The response obtained from H43 N125 is short in duration but has nearly the same maximum amplitude as the H43 response and much faster time course of rise than the H43 response. The shortness of response duration may be due to the fact that, under the conditions of this experiment (restricted DAG generation), the response cannot be sustained during a bright prolonged stimulus. This response arises as a result of DAGL activity because further reducing the DAGL activity (xl18 mutation) abolishes the response. However, the fact that adding N125 to the H43 background resulted in a response of faster time course may also be a manifestation of the enhancing and facilitatory effects of DAG on the excitatory agent. Speculating further, the facilitatory action of DAG might be in place to ensure that the excitation of channels is light regulated, because DAGL activity is not light regulated while the generation of DAG is (Leung, 2008).
The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. This study identified two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits (Rosenzweig, 2008).
As has been reported using RNAi, this study confirmed, using classical genetic mutations, that dTRPA1 is essential for larval warm avoidance. These findings were extended to show that cool avoidance does not require dTRPA1. Rather, it was discovered that cool avoidance depends on the TRPC family members TRPL and TRP. Although these two TRP channels also have critical functions in phototransduction, larval cool avoidance and phototransduction are distinct, since neither larval photoreceptors nor the phototransduction molecules NORPA and INAF are required for cool avoidance (Rosenzweig, 2008).
TRPL and TRP belong to the TRPC family of TRP channels. The TRPC family is evolutionarily conserved, with seven members in mammals. Although individual members of the TRPV, TRPM, and TRPA families are known to be temperature-activated ion channels, an involvement for TRPC proteins in mediating temperature perception has not been previously demonstrated, and it will be interesting to learn whether mammalian TRPCs also contribute to thermosensation. This study demonstrates a clear requirement for the Drosophila TRPCs TRPL and, to a lesser extent, TRP in cool avoidance. However, in contrast to classic thermoTRPs such as dTRPA1, which exhibits strong warmth activation when ectopically expressed in oocytes, neither TRPL nor TRP showed detectable cool activation in oocytes. In addition, whereas expression of TRPL and TRP was readily detected in the larval photoreceptors using RNA in situ hybridization, expression could not be detected in the putative cold receptor neurons of the terminal organ. Thus, whether these TRPC proteins participate directly in thermotransduction or affect thermosensory behavior by acting at a downstream step remains to be determined. Although the mechanism by which TRPL and TRP mediate cool avoidance is not clear, it appears distinct from the mechanisms by which TRPL and TRP channels mediate visual system signal transduction, since the latter rely on NORPA and INAF, which are dispensable for cool avoidance (Rosenzweig, 2008).
It was also found that that the neural pathways for cool and warm avoidance are distinct. Whereas the larval cold sensors, located in the terminal organ, are essential for larval cool avoidance, they are not necessary for warm avoidance. As for the larval warm sensors, a set of dTRPA1-expressing neurons in the brain has been implicated in warm avoidance in third instar larvae. Unfortunately, it is not yet technically possible to assess the function of these cells in cool avoidance, since available promoters for manipulating these dTRPA1-expressing neurons are expressed too late to effectively manipulate neuronal function in first and second instar larvae, the stages at which cold avoidance is most robust (Rosenzweig, 2008).
Together these data indicate that Drosophila use distinct TRP channels and neurons to respond to different, discrete ranges of temperature. The channels TRPL and TRP and the neurons of the terminal organ are specifically involved in the avoidance of cool temperatures, whereas dTRPA1- and dTRPA1-expressing neurons are required for the avoidance of moderately warm temperatures. At even higher temperatures, Painless mediates avoidance by acting in multiple-dendritic neurons, whereas Pyrexia has a potentially general neuroprotective effect possibly reflecting its broad neuronal expression. Thus, Drosophila possesses a suite of thermosensory detection pathways, each of which responds at specific temperatures and promotes a characteristic set of behavioral responses, ranging from gradual migration away from moderately warm or cool temperatures to immediate withdrawal from extreme temperatures that cause rapid tissue damage. As mammals also use distinct sensors for detecting different portions of the thermal spectrum, these studies support a fundamental similarity in the logic of thermosensation in both mammals and insects, with both types of animals sensing the range of temperatures they encounter using a series of TRPs and thermosensory cells, with different sensors tuned to different portions of the temperature spectrum (Rosenzweig, 2008).
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date revised: 15 August 2009
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