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Photoreceptor Degeneration and Ca2+ Influx through Light-Activated Channels of Drosophila

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We discuss in this chapter the role of Ca2+ homeostasis in maintaining the structural integrity of photoreceptor cells in Drosophila. Both insufficient and excessive amounts of Ca2+ in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels in Drosophila photoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+ homeostasis. We will begin by reviewing Drosophila phototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+ entry through light-sensitive channels and the presumed mechanisms by which too little and too much Ca2+ entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+ entry through light-sensitive channels, leading to massive photoreceptor degeneration.

Invertebrate Phototransduction

Much has been learned about the phototransduction process in Drosophila in recent years (reviewed by, e.g. Pak1; Zuker2; Montell3; Minke and Hardie4). The phototransduction pathway is based on a phosphoinositide-mediated signaling cascade, in which photoexcited rhodopsin activates a phospholipase Cβ (PLC) through the activation of a G protein, Gq. The central role PLC plays in this process is demonstrated by the fact that no light-induced response can be elicited from severely affected mutants of the PLC-encoding norpA gene.5,6 Activation of phospholipase C leads to the opening of two classes of phototransduction channels: TRP and TRPL channels, encoded, totally or in part, by the trp (transient receptor potential) and trpl (trp-like) genes,7,8,9,10 respectively. The TRP channel is widely considered to be homomultimeric, consisting only of TRP as subunits. The subunit composition of the TRPL channel, however, is still under debate.3,4 Evidence has been presented both for the model that the TRPL channel consists only of TRPL protein as subunits11,12,13 and the model that it is heteromultimeric, consisting of both TRP and TRPL proteins as subunits.14 However, a double mutant lacking both the TRP and TRPL proteins is totally unresponsive to light,12,13 suggesting either that each channel is a homomultimer consisting only of TRP or TRPL or that the TRP and TRPL proteins constitute functionally indispensable portions of the respective channels.

Although the identity of these channels has been known for some time, the elucidation of the mechanisms of their activation and regulation proved elusive. In other phosphoinositide-mediated signaling systems, PLCβ, when activated, is known to hydrolyze the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to generate two potential second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (review: Berridge15). IP3 binds to the IP3 receptor (IP3R) to release Ca2+ from internal stores, while DAG is thought to activate protein kinase C. One of the earliest hypotheses to be put forth on Drosophila phototransduction was that IP3 generated in the hydrolysis of PIP2 by PLC binds to IP3 receptors on internal stores and the ensuing depletion of internal Ca2+ stores signals the opening of the TRP channels on the plasma membrane (Minke and Selinger16). However, it was difficult to find definitive experimental support for the hypothesis. For example, depleting internal stores with thapsigargin, an endoplasmic Ca2+ ATPase inhibitor, does not activate light-sensitive channels in Drosophila photoreceptors (Ranganathan,17 Hardie18). The most damaging piece of evidence against this hypothesis is that null or near-null mutations in the only known IP3 receptor gene of Drosophila are found to have no effect on phototransduction (Acharya,19 Raghu20). Unless there is another IP3 receptor gene that has not yet been identified by the Drosophila Genome Project, the above finding appears to rule out participation of the IP3 branch in Drosophila phototransduction. Moreover, Chyb21 found that both TRP and TRPL channels could be activated by polyunsaturated fatty acids (PUFAs). Since PUFAs can be generated from DAG by DAG lipase, the above finding seems to suggest that the DAG branch, rather than the IP3 branch, of the phosphoinositide cascade is utilized in Drosophila phototransduction.

The final chapter apparently has not been written on this story, however. Two recent papers challenged the idea that the DAG branch is involved in phototransduction. In one of these, Chorna-Ornan22 reported that a newly discovered membrane-permeant IP3 receptor antagonist, 2-APB (2-aminoethoxydiphenyl borate) was found to reversibly block responses of Drosophila photoreceptors to light, suggesting that the IP3 branch may play a role in phototransduction after all. In the other paper, Agam23 reported that metabolic stress, such as anoxia, mitochondrial uncoupling, and ATP depletion, reversibly activates TRP and TRPL channels. They further pointed out that PUFAs are efficient mitochondrial uncouplers (Arslan24; Hermesh25) and suggested that the previously reported activation of TRP and TRPL channels by PUFAs may be an indirect result of mitochondrial uncoupling. It seems clear additional research is needed to sort out these conflicting findings.

Ca2+Entry through TRP and TRPL Channels

Whole cell recordings of the light-induced current (LIC) from photoreceptors in dissociated ommatidia showed that in wild-type flies, in which both the TRP and TRPL channels are functional, the reversal potential of LIC is strongly dependent on extracellular concentration of Ca2+, suggesting that the light-activated channels are highly Ca2+ permeable (PCa:PNa of ˜24:1) (Hardie26). Subsequently, Hardie and Minke9 showed that the LIC in wild-type is mediated by two distinct classes of light-activated channels, TRP and TRPL, and that the TRP channel is highly Ca-permeable (PCa:PCs of >100:1)(Reuss13), while the TRPL channel is nonspecifically cation selective but with a significant Ca2+ permeability (Peretz27; Hardie18), the PCa:Pcs ratio being about 4 (Reuss13). Ca2+ ions entering through these channels regulate the current through the two classes of channels differentially by feedback (Hardie26; Reuss13). The current through the TRP channel is facilitated first, causing a rapid increase in current influx, and then rapidly inhibited. The current through the TRPL channel is also inhibited but without the initial facilitation. Because only the TRP channel is initially facilitated under physiological conditions, most of the current entering the cell in response to light does so through the TRP channel (Reuss13).

Magnitude of Ca2+Entry and Photoreceptor Degeneration

Photoreceptors do not seem to tolerate well either insufficient or excessive amounts of Ca2+ in the cell. The mutations, rdgA and TrpP365, have been shown recently to cause an excessive entry of Ca2+ into photoreceptors by rendering TRP channels constitutively active.28,29 Cytotoxic effects of Ca2+ are well-documented,30,31 and massive degeneration of photoreceptors ensues as a consequence of the constitutive activity. The degeneration caused by these mutations is among the most severe and earliest-onset reported for Drosophila. It can proceed in the absence of light, though light seems to further exacerbate the course of degeneration.29 These mutations are discussed in some detail in the next sections.

Insufficient Ca2+ has been reported to cause light-dependent, late-onset photoreceptor degeneration by interfering with recycling of the rhodopsin inhibitor molecule, arrestin.32 Following photoactivation of rhodopsin to metarhodopsin, inactivation of metarhodopsin begins almost immediately. As in vertebrates, inactivation of metarhodopsin involves arrestin binding.33,34 Unlike in vertebrates, however, binding of Drosophila arrestins to metarhodopsin is not dependent on metarhodopsin phosphorylation.35,36The metarhodopsin-bound arrestin is then phosphorylated by Ca2+-calmodulin dependent kinase II37 to allow its release from metarhodopsin binding.38 If arrestin is not phosphorylated or insufficiently phosphorylated, its release from metarhodopsin is inhibited, and metarhodopsin-arrestin complexes accumulate.38,36 The complexes are then internalized by endocytosis, and this endocytosis appears to trigger apoptosis of photoreceptor cells by unknown mechanisms.32,36 Thus, any mutation that substantially decreases Ca2+ entry through the light-activated channels is expected to cause degeneration through inefficient phosphorylation of arrestin.32 It has long been known that many Drosophila mutations that affect phototransduction also cause photoreceptor degeneration.39–45 Since these mutations affect the phototransduction process, many of them are expected to cause substantially reduced Ca2+ influx through the TRP channels. Light-dependent degeneration of a relatively slow time course observed in many of these mutants may have its origin in the above mechanism. Indeed, Alloway32 showed that in two such mutants, rdgB and norpA (The rdgB gene encodes a phophatidylinositol transfer protein, presumably involved in membrane transport of phospholipids needed for phototransduction (Vihtelic46), and the norpA gene encodes a phospholipase Cβ, which plays a central role in Drosophila phototransduction (Bloomquist6), photoreceptor degeneration appears to result from the formation of stable metarhodopsin-arrestin complexes. In the next sections, we will discuss trp and inaF null and near-null mutants, in which most of the TRP channels are either absent or nonfunctional. It is possible that light-dependent, slowly progressing degeneration observed in these mutants also may have the same origin.

Degeneration Caused by Constitutive Activity of the TRP Channel

In this section we discuss the mutants rdgA and TrpP365, both of which display severe photoreceptor degeneration shown to be due to constitutive activity of the TRP channel.


rdgA (retinal degeneration A) mutants were first isolated in 1970 by behavioral assays for nonphototaxis by Hotta and Benzer.47 Shortly thereafter, additional rdgA mutant alleles were recovered in other laboratories using somewhat different methods (review: Pak48). In severely affected rdgA mutants, degeneration is well advanced already at eclosion (Johnson49; Matsumoto50). Although degeneration proceeds either in the presence or absence of light, illumination appears to accelerate the time course of degeneration (K. Isono and Pak, unpublished observation). Biochemical studies first provided evidence that rdgA mutants are deficient in the activity of diacylglycerol kinase (DGK),51–53 an enzyme that catalyzes the phosphorylation of diacylglycerol (DAG) to convert it to phosphatidic acid (PA) in the first step of resynthesis of phosphatidylinositols. The rdgA gene was cloned by Masai54and shown to encode an eye-specific DGK.

The reason for the degeneration, however, remained obscure until Raghu28 subjected rdgA mutants to whole cell, patch clamp recordings. The most significant finding to emerge from this study is that all rdgA photoreceptors on which recordings were made (n = 45) display a small (˜50 pA), noisy, constitutive inward current, which is detectable immediately upon establishing the whole cell configuration. The following lines of evidence established that the TRP channels are primarily responsible for the constitutive current.

  1. The current was blocked by La3+, which is known to block TRP-mediated current (Hochstrate55; Suss56; Hardie and Minke9).
  2. The I-V relationship, which plots current against the membrane voltage, closely resembled that of the TRP conductance.
  3. The high frequency noise characteristics (power spectra) were indistinguishable between the constitutive current and the TRP-mediated current.

Eliminating the TRP channels in rdgA mutants by constructing rdgA;;trp double mutants rescued the severe early-onset degeneration. The rescue was specific to the elimination of the TRP channels because eliminating the TRPL channels by constructing rdgA;; trpl double mutants did not rescue the early degeneration phenotype. There was, however, also slower degeneration of photoreceptors in rdgA;;trp double mutants with a time course of 2–6 weeks, and this slow degeneration was still present when the TRPL channels were also eliminated, i.e., present in rdgA;trpl;trp triple mutants. This slow degeneration is also present in strong trp mutants (see next section) and may represent degeneration due to insufficient Ca2+ in the cell, mentioned in the previous section.

The above results suggested that the early-onset degeneration in rdgA is caused by an uncontrolled Ca2+ influx through the TRP channels. The problem with this interpretation was that the constitutive current was still present in rdgA;;trp double mutants, even though the early-onset degeneration phenotype had been essentially eliminated. Unlike in rdgA, however, the constitutive current in the double mutants was carried by the TRPL channels because (1) it was not blocked by La3+, (2) its I-V relationship and noise power spectra were similar to those of TRPL channel currents, and (3) it was no longer present in rdgA;trpl;trp triple mutants, i.e., eliminating the TRPL channels eliminated the current. Since the TRPL channels also mediate substantial Ca2+ fluxes,27,18 it would be difficult to explain the rescue of early-onset degeneration in rdgA;;trp, but not in rdgA;trpl, if uncontrolled influx of Ca2+ were responsible for the degeneration.

To resolve this paradox, these authors compared the developmental onsets of constitutive activity and early signs of degeneration at the pupal stage. They found that TRP channels become constitutively active somewhat earlier than the TRPL channels (˜70 hr vs. 85–90 hr of the pupal stage). Electrophysiologically detectable early signs of degeneration seemed to occur at around 75–80 hr in rdgA mutants, coinciding with the first appearance of constitutive TRP channel activity in these mutants. Raghu28, therefore, suggested that there is a window of Ca2+ sensitivity for degeneration at ˜75–85 hr of the pupal stage and that photoreceptors become resistant to Ca2+ past this window. Thus, they argued, while both the TRP and TRPL channels become constitutively active in rdgA, the constitutive activity of the TRP channels occurs somewhat earlier than the TRPL channels, and the uncontrolled Ca2+ through the TRP channels at this earlier time window is responsible for the early-onset photoreceptor degeneration in rdgA.

Chyb21 had shown earlier that polyunsaturated fatty acids (PUFAs) could activate both TRP and TRPL channels in Drosophila photoreceptors. DAG can be metabolized via two potential pathways: (1) by DAG lipase to generate PUFAs or (2) by diacylglycerol kinase (DGK) to be converted to phosphatidic acid (PA). In rdgA mutants, conversion of DAG to PA is blocked, and, moreover, DAG levels are reported to be normal (Inoue53), suggesting enhanced metabolization of DAG via the DAG lipase pathway. Therefore, excess amounts of PUFAs would be generated resulting in constitutive activities of TRP and TRPL channels. Raghu28 thus argued that their results with rdgA provided independent genetic support for the proposal that PUFAs may be messengers of excitation in Drosophila photoreceptors.


TrpP365 is a newly identified mutation in the trp (transient receptor potential) gene that causes constitutive activity of the TRP channel.29

The first mutant isolated in the trp gene was a naturally occurring one that behaved as if blind in bright ambient light.57,58 Subsequently, other recessive mutations of this gene were isolated through chemical mutagenesis.59 Both ERG (electroretinogram: extracellularly recorded light-evoked mass response of the eye)57 and intracellular60 recordings revealed that the photoreceptor potential is much smaller than the wild-type potential and decays to baseline during a bright and/or prolonged light stimulus (Fig. 1D), thus the name trp. In contrast, in wild-type a steady-state or maintained component is present throughout the duration of the stimulus (Fig. 1A). In addition to the electrophysiological phenotype, null or near-null trp mutants display late-onset, light-dependent photoreceptor degeneration (see Fig. 4).61,62,63 This phenotype is discussed further below.

Figure 1. Representative electroretinograms (ERGs) of wild-type, classical trp mutants, and mutants carrying TrpP365 allele, showing that TrpP365 causes a mutant phenotype distinct from that of the classical trp mutants.

Figure 1

Representative electroretinograms (ERGs) of wild-type, classical trp mutants, and mutants carrying TrpP365 allele, showing that TrpP365 causes a mutant phenotype distinct from that of the classical trp mutants. a) wild type, b) TrpP365 heterozygote, c) (more...)

Figure 4. Time course of photoreceptor degeneration in TrpP365 hterozygotes, trp near-null mutants, and inaF null mutants, and mutual suppression of degeneration by inaF and TrpP365.

Figure 4

Time course of photoreceptor degeneration in TrpP365 hterozygotes, trp near-null mutants, and inaF null mutants, and mutual suppression of degeneration by inaF and TrpP365. The degeneration time courses were determined by monitoring the disapearance of (more...)

The trp gene was cloned independently by Montell and Rubin7 and Wong8. As discussed previously (sections on Phototransduction and Ca2+ entry), the gene encodes the subunits of the highly Ca2+-permeable class of light-sensitive channels, TRP. Protein (Western) blot analyses showed that the quantity of the TRP protein in null or near-null trp mutants is reduced to undetectable amounts. Thus, in trp null or near-null mutants, the amount of Ca2+ entering the photoreceptor cells in response to light is greatly reduced.64,27,18 Insofar as insufficient Ca2+ in photoreceptor cells can be a cause of photoreceptor degeneration, the late-onset, slowly progressing degeneration seen in null or near-null trp mutants may be due to insufficient Ca2+ entry (see section on Magnitude of Ca2+ entry and photoreceptor degeneration).

The TrpP365 mutant was isolated in chemical mutagenesis. It has phenotypes very distinct from those of null and near-null trp mutants described above.29 Its most conspicuous phenotype is the early-onset, very rapid, and massive photoreceptor degeneration (Fig. 2). The ERG is nearly absent in homozygotes, but the response remaining is not transient but lasts the entire duration of stimulus (Fig. 1C). Moreover, the mutation does not seem to affect the amount of the TRP protein directly. Whatever decrease in the amount of the TRP protein observed appears to be attributable to photoreceptor degeneration. Finally, the mutation is semi-dominant for all these phenotypes in that the phenotypes are observed in heterozygotes as well as in homozygotes although phenotypes of heterozygotes are less severe than those of homozygotes (Figs. 1B, 1C, & 2).

Figure 2. Electron micrographs showing severe photoreceptor degeneration caused by TrpP365 and exacerbation of degeneration by exposure to light.

Figure 2

Electron micrographs showing severe photoreceptor degeneration caused by TrpP365 and exacerbation of degeneration by exposure to light. Transverse sections of retinas were obtained near the R7 and R8 rhabdomere bounderies from TrpP365 homozygotes raised (more...)

Despite this very different set of phenotypes displayed by TrpP365 in comparison to all other known trp mutants, the following lines of evidence unequivocally established that mutation(s) in the trp gene is responsible for the TrpP365 phenotype:29

  1. nucleotide substitutions were identified in the trp coding region of the TrpP365 mutant, which would cause alterations of amino acid sequence in the TRP protein;
  2. a transgene containing the trp gene isolated from the TrpP365 mutant induced the mutant ERG phenotype in a wildtype background in a dose-dependent manner; and
  3. a transgene containing the wild type trp gene partially rescued the mutant phenotype in the TrpP365 background in a dose-dependent manner.

Patch clamp wholecell recordings were carried out on photoreceptors in dissociated ommatidia of TrpP365 in an attempt to determine the cause of degeneration.29 The results showed that in TrpP365 homozygotes, but not in wild type, outwardly rectifying membrane currents were detected, as soon as the whole cell configuration was established, in response to membrane voltage steps in the absence of light stimulus. Applying La3+, which blocks the TRP channel but not the TRPL channel (Hochstrate55; Suss56; Hardie and Minke9; Niemeyer11), abolished the currents. Thus, it appears that the TRP channel is constitutively active in TrpP365 mutants, and the consequent excess Ca2+ entry into the photoreceptors appears to be responsible for the observed early-onset, rapid degeneration.

Sequence analysis showed that TrpP365carries four mutations that would alter the TRP protein sequence.29 To determine which of these four might be responsible for the TrpP365 mutant phenotypes, transgenic flies, each carrying either one of the four mutations singly or three or more in various multiple combinations, were generated and tested for their ERG and degeneration phenotypes. Results showed that the primary cause of the TrpP365 mutant phenotypes is an alteration of the 550th amino acid residue, phenylalanine, to isoleucine (Hong et al, submitted). The TRP channel has some homology to voltage-gated Na and Ca2+ channels,10 and the Phe550 is located near the N-terminal end of the fifth transmembrane domain. The above results suggested that Phe550 might be important for the gating or regulation mechanism(s) of the TRP channel.

The mechanisms of the TRP channel activation and regulation are not understood (see Phototransduction section). We have hypothesized that additional, still unidentified proteins might be involved in the activation and regulation processes. One possible approach to identifying such proteins is to search for mutants with phenotypes similar to that of trp and then to identify the proteins corresponding to the mutants. The rationale for this approach is that any defect in the proteins involved in the activation or regulation of the TRP channel is likely to cause the TRP channel function to be defective. inaF mutants are among the first such mutants to be identified and characterized.62The first inaF mutant was generated by P element insertional mutagenesis, and several other inaF alleles, including null alleles, were recovered subsequently through imprecise excision of the P element from the original inaF mutant. The receptor potential of a null mutant, inaFP106x, decays to the baseline during bright light stimulus, in a manner very similar to that of null or near-null trp mutants. Furthermore, inaFP106x displayed photoreceptor degeneration with an onset and kinetics approximating that of a near-null trp mutant (Fig. 4 62).

The similarity of the ERG phenotype between inaFP106x and null or near-null trp mutants suggested that the TRP channels are only marginally functional in the null inaF mutant as in near-null trp mutants. To compare the effects of inaF and trp mutations on the TRP channel function, the TRPL channels were genetically eliminated by introducing a null trpl mutation, trpl302, into the inaFP106x or trpP301 background, i.e., by constructing the trpl302;trpP301 and inaFP106x;trpl302 double mutants, where trpl302 and inaFP106x are null alleles and trpP301 is a near-null allele. In trpl302, which still has a full complement of TRP channels and used as a positive control, a light stimulus produces a robust response that lasts the entire duration of illumination (Fig. 3A). In trpl302;trpP301 and inaFP106x; trpl302 double mutants, on the other hand, the responses are severely reduced in size and duration in a very similar manner (Fig. 3A). Thus, the effect of inaFP106x on the TRP channel is very similar to that of trp,P301 which reduces the TRP concentration to undetectable levels. The effect is specific for the TRP channel because inaFP106x has little or no effect on the TRPL channel (Fig. 3B). Western analysis revealed that the quantity of the TRP protein is drastically reduced in the inaFP106x mutant to approximately 6% of that of wild type. This reduction appears to be specific for TRP because the reduction is not observed in three other retinal proteins Rh1, INAD and PLC that are known to function in the phototransduction pathway.62 These results suggested that a part of the inaFP106x phenotype might be due to the reduction in quantity of the TRP protein. It should be noted, however, that in trpP301, to which inaFP106x is being compared, the TRP protein is reduced to an undetectable level in Western analysis.

Figure 3. Effects of inaF and trp mutations on the photoreceptor responses generated through the TRP a) or TRPL b) channels.

Figure 3

Effects of inaF and trp mutations on the photoreceptor responses generated through the TRP a) or TRPL b) channels. In a), the TRP channels were isolated using a trpll null mutation, and the effects of inaF null mutation and trp null and near-null mutations (more...)

Sequence analysis showed that the inaF gene encodes a novel protein inaF of 241 amino acid residues. inaF is devoid of any trans-membrane domains and contains no obvious domains or motifs. Database search has not resulted in the identification of any known proteins with significant sequence similarity to inaF.

The function of the inaF protein has not yet been determined. There are at least three viable hypotheses. One is that inaF is required for the gating of the TRP channels62 and, without it, the TRP channels cannot open effectively upon light stimulus. The reduction in the quantity of the TRP protein may reflect secondary effects resulting from the absence of inaF. An alternative hypothesis is that inaF is required for maintaining the quantity of TRP protein either by enhancing the synthesis or preventing the degradation of the TRP protein. The apparent trp-like phenotype may reflect dearth of functional TRP protein. The third possibility is the combination of the above two, i.e., inaF is required for both the gating of the TRP channels and the maintenance of the quantity of the TRP protein. While experimental support can be found for all three, none is as yet definitive.

Regardless of the actual mechanism(s) involved, the net effect of inaF mutations is to make the TRP channels ineffective in mediating membrane currents. If the TrpP365 mutation causes the TRP channel to become constitutively active and a null or near-null inaF mutation renders the TRP channel ineffective, it seemed possible that the two mutations might compensate for each other's effects. Accordingly, the double mutant, inaFP106x;;TrpP365/+, homozygous for inaFP106 and heterozygous for TrpP365 was constructed and tested for its phenotypes. The results showed that the two mutations, indeed, suppress the effects of each other. (Suppression was also present in double mutants involving TrpP365 homozygotes, but was more clearly seen in ones involving heterozygotes.) Illustrated in Fig. 4 are the retinal degeneration time courses, as monitored by the deep pseudopupil (dpp), of the double mutant, inaFP106x;;TrpP365/+, and the parental stocks, inaFP106x and TrpP365/+, as well as trpP301. The dpp is a superposed virtual image of a group of neighboring rhabdomere tips observed in a living fly using a dissecting microscope (Franceschini65). When the superposition fails, the dpp disappears. Thus, dpp is a sensitive indicator of not only the disappearance but also the disarrangement of the rhabdomeres resulting from degeneration. Because of its sensitivity, the degeneration time course determined by dpp generally tends to be faster than that determined by anatomical analyses. In TrpP365 heterozygotes, the dpp starts disappearing almost immediately after eclosion and is completely gone by 5 days posteclosion. This fast dpp disappearance represents the rapid degeneration due to constitutive activity of the TRP channel. In trpP301 as well as inaFP106x, the rapid degeneration is not present, but there is a slower disappearance of dpp that begins around 5–6 days post-eclosion. This slow disappearance represents the slowly progressing, late-onset degeneration normally seen in trp and inaF null and near-null mutants, and may be due to insufficient Ca2+ influx (see Section on Magnitude of Ca2+ entry). In the double mutant, inaFP106x;;TrpP365/+, the rapid disappearance of dpp was no longer present, and even the slower dpp disappearance has greatly slowed in time course and improved in severity. We interpret these results to suggest that in the double mutant,

  1. the early-onset degeneration due to TrpP365 is no longer present because of the restricted Ca2+ entry due to the presence of the inaFP106x mutation, and
  2. the late-onset degeneration is also much improved because TrpP365 mutant channels allow some Ca2+ to enter the cell even in the presence of inaFP106x.

These results are consistent with the idea that the slowly progressing late-onset degeneration of trp and inaF null and near-null mutants may indeed be due to insufficient Ca2+ entry and suggest, furthermore, the importance of the inaF protein in TRP channel function and Ca2+ homoeostasis in photoreceptor cells.


Perturbed Ca2+ influx appears to be one of the major causes of photoreceptor degeneration in Drosophila. Excessive Ca2+ influx through the TRP channels causes severe, early-onset photoreceptor degeneration seen in such mutants as rdgA and TrpP365. On the other hand, a drastic reduction in the TRP channel activity, as in the null or near-null trp and inaF mutants, causes photoreceptor degeneration of lateonset and slow progression.

In the double mutant, inaFP106x;;TrpP365/+, the early-onset degeneration seen in TrpP365/+ is eliminated and the late-onset, slowly progressing degeneration seen in inaFP106x is greatly ameliorated. Because TrpP365 and inaFP106x have opposing effects on Ca2+ influx, the rescue of the degeneration phenotypes may be due to restoration of appropriate Ca2+ influx through the TRP channels.


Work done in the authors' laboratory was supported by a grant from the National Institutes of Health, National Eye Institute (EY 00033) to WLP. We thank Sara Dykes for help in preparation of the manuscript.


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