The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. These channels are Ca²⁺ permeable and have been implicated as an important component of cellular Ca²⁺ homeostasis in neuronal and nonneuronal cells. The power of the molecular genetics of Drosophila has yielded several mutants in which constitutive activity of TRP leads to a rapid retinal degeneration in the dark. Metabolic stress activates rapidly and reversibly the TRP channels in the dark in a constitutive manner by a still unknown mechanism. The link of TRP gating to the metabolic state of the cell is shared also by mammalian homologues of TRP and makes cells expressing TRP extremely vulnerable to metabolic stress, a mechanism that may underlie retinal degeneration and neuronal cell death.

Introduction

The visual system of Drosophila melanogaster¹ has been the subject of genetic analysis through isolation of visual mutants² and application of molecular genetics combined with powerful electrophysiological and biochemical functional tests (for review see refs. 1–5). One of the key proteins that have been discovered due to the power of the Drosophila genetics is the protein designated by Minke Transient Receptor Potential (TRP) because of the unique phenotype of the trp mutant.⁶ While genetic evidence has unequivocally demonstrated that phospholipase C is absolutely required for light excitation in Drosophila photoreceptors,^2,7 a major unresolved question of Drosophila phototransduction is the gating mechanism of light-sensitive channels TRP and TRP-like (TRPL) and the identity of the second messenger of excitation.¹

Photoreceptor degeneration is a phenomenon, which appears in strong alleles of almost every phototransduction-defective mutant of Drosophila. Given the abundance of the signaling molecules in the microvilli (the signaling membranes of the photoreceptor cell) and the crucial roles of these molecules in the assembly and function of the phototransduction machinery, mutants defective in the signaling proteins are likely to show degeneration.¹ Because of the great interest in neuronal cell death in general and in human retinal degeneration, a search for mammalian homologues of Drosophila genes has been recently pursued with much effort. Indeed, mammalian homologues of the Drosophila genes rdgB,^8,9 rdgC,^10,11 ninaC¹² and trp¹³ have been found in mammalian retina. These genes cause photoreceptor degeneration in Drosophila upon malfunction. However, the function of these molecules in vertebrate cells in not clear. Despite extensive investigation, relatively little is known about the molecular mechanisms underlying retinal degeneration due to mutations in the above genes also in invertebrate species.

It has been recently found that the TRP channel is involved in retinal degeneration of several Drosophila mutants in which constitutive activity of TRP seems to cause extremely rapid retinal degeneration.^14,15 Therefore, the phenomenon of constitutive activity of TRP and TRPL channels in the dark and the ensuing influx of Ca²⁺, which possibly lead to retinal degeneration are the main topics of this review.

The Drosophila TRP and TRPL Channels

The Molecular Structure of Drosophila TRP and TRPL

The trp gene was cloned by Montell and Rubin¹⁶ and by Wong and colleagues.¹⁷ The molecular structure of the TRP protein shows multiple domains that are likely to play an important role in cellular functions.³ TRPL was isolated using a screen to detect calmodulin binding proteins and shared overall 40% identity with TRP with much greater similarity (∼70%) in the putative transmembrane regions.¹⁸ The N- and C-termini of TRP and TRPL both contain a number of recognizable motifs, but their function in TRP and TRPL is unknown (Fig. 1). Both TRP and TRPL have weak but significant homologies to a known channel subunit of vertebrate voltage-gated Ca²⁺ channel (the dihydropyridine receptor). By analogy to voltage-gated K channels and the cyclic nucleotide-gated channels, both trp and trpl gene products represent subunits of putative tetrameric channels. Since null trp or trpl mutants both respond to light, each can clearly function without the other. However, heterologous co-expression studies and co-immunoprecipitation led to the suggestion that in wild-type (WT) flies the light-induced conductance was composed of TRP or TRPL homomers and TRP-TRPL heteromultimers.¹⁹ Detailed in situ measurements of biophysical properties including ionic selectivity and single channel conductance questioned this conclusion.²⁰ A recent study has shown that there is an additional subunit(s) called TRPγ,²¹ which is highly enriched in the photoreceptor cells. The N-terminal domain of TRPγ dominantly suppressed the TRPL-dependent transient receptor potential on a trp mutant background, suggesting that TRPL-TRPγ heteromultimers contribute to the photoresponse. Furthermore, TRPL and TRPγ coimmunoprecipitate, suggesting that physical interaction between these proteins forms heteromeric channels.²¹

Figure 1

Putative domain structure and topology of Drosophila TRP. TRP contains six putative transmembrane helices S1-S6 and a putative pore region between S5 and S6, S3-S6 show sequence fragments identical to the equivalent regions of the dihydropyridine receptor (more...)

The TRP Family of Channel Proteins

Constitutive Activity of the TRP Channel by Mutations and by Metabolic Stress May Underlie Retinal Degeneration

Mutations in the Transmembrane Domain of TRP Cause Rapid Photoreceptor Degeneration

Our understanding of TRP function and regulation has been augmented in the past by the availability of flies mutated in trp. However, especially informative should be flies that possess a TRP protein altered in its function. unfortunately, the known Drosophila trp mutants either completely lack TRP protein or have a highly reduced amount of functionally normal TRP. Furthermore, attempting to directly mutate the trp gene itself, by way of systematic site-directed mutagenesis, failed to yield any significant information on TRP function or structure. This is because mutated proteins were either unstable or not expressed at all in transgenic flies (leading to a full null phenotypes) or, alternately, the flies exhibited a completely wild-type phenotype, implying that those modifications had no phenotypic consequences (unpublished results). Recently, Pak and colleagues have identified a novel trp mutant (named Trp^P365), which genetically mapped to the trp locus and has three mutations in the transmembrane domain of TRP.¹⁵ Trp^P365 seems to be the first trp allele that expresses a modified (yet presumably active; see below) TRP protein. Phototransduction in Trp^P365 mutant flies is aberrant, however; instead of displaying the classical trp null phenotype (a receptor potential that declines to the dark level during illumination) the Trp^P365 mutation leads to a total absence of light response, with a constitutive current in the dark (see below). Strikingly, unlike any other trp alleles, these mutants also show extremely rapid retinal degeneration. Thus, the Trp^P365 mutant provides us with a first and unique opportunity to characterize essential mechanisms underlying the physiological function of TRP and the TRP-dependent light-activated conductance.¹⁵

Functional Analysis at the Single Cell Level by Whole-Cell, Patch-Clamp Recordings Revealed Constitutive Activity of the TRP Channels

Whole-cell voltage clamp recordings were performed on R1-6 photoreceptors in dissociated ommatidia preparations of Trp^P365 homozygotes, Trp^P365 heterozygotes, and wild type. The light-induced current (LIC) of the heterozygote Trp^P365/+ was indistinguishable from that of wild-type flies. This was not true for most of the ommatidia of the heteroallele Trp^P365/Trp^CM. In contrast, Trp^P365/Trp^P365 homozygote did not respond to light of any intensity. Large fraction (20%–70%) of the Trp^P365/Trp^CM heterozygote ommatidia did not respond to light, like the Trp^P365/Trp^P365 (Fig. 2A). The LICs of Trp^P365/Trp^CM flies revealed abnormal features such as highly reduced sensitivity to light and abnormally slow kinetics.

Figure 2

Single-cell functional analysis by whole-cell recordings showing constitutive activity of the TRP channel in the Trp^P365/Trp^P365 and Trp^P365/trp^CM mutants. Panel A shows a typical light-induced current (LIC) of a wild-type cell (left trace) in response (more...)

To investigate the reason for the inability of cells to respond to light, after the establishment of whole-cell recordings we stepped the holding voltage to different membrane potentials in the range between +80 mV to −120 mV in steps of 20 mV in the dark. In WT cells no significant currents were observed. In contrast, in the nonresponsive mutants, at positive membrane potentials large outward currents were recorded with strong outward rectification (Fig. 2B). The constitutive currents, which were recorded in a large fraction of Trp^P365/Trp^CM heterozygote cells and in all Trp^P365/Trp^P365 cells, were very similar to the current, which reflects activation of the TRP channels of wild-type cells during light. However, in sharp contrast to wild-type photoreceptors, the few Trp^P365/Trp^P365 cells from which recordings could be made and in all light-insensitive Trp^P365/Trp^CM cells, voltage steps elicited outward currents in the dark from the moment the recording configuration was established.

The constitutive current of both homozygote and heterozygote Trp^P365 cells had all the properties of the TRP-dependent current: In Ca²⁺-containing medium large outward currents were observed in the homozygote and heterozygote Trp^P365 cells, and these currents were very similar to these of WT cells under continuous light conditions. In addition, application of 10 μM La³⁺ to the extracellular medium blocked the TRP-dependent current and the constitutive currents in a very similar manner (Fig. 2C). Since these currents are mediated by the TRP channel, the TRP channel in these photoreceptors appears to be already open at the time the recording configuration is being established in the nonresponding cells of the Trp^P365/Trp^CM heterozygote and in all Trp^P365/Trp^P365 cells but not in WT ommatidia.

The above study thus shows that the Trp^P365 mutation highly increased the probability of the TRP channels to open in the dark in correlation to the Trp^P365 dosage. Strikingly, constitutively activity of TRP channels was recorded even in cells that do not show any sign of morphological degeneration (i.e., in Trp^P365/Trp^CM heterozygote cells), thus suggesting that the constitutive activation of the channels precedes the degeneration.¹⁵

Metabolic Stress Constitutively Opens the TRP and TRPL Channels in the Dark at a Late Stage of the Cascade

Anoxia is known to rapidly and reversibly depolarize the photoreceptor cells of the fly in the dark, possibly via openings of the light-activated channels. It is well-established that Ca²⁺ influx into the photoreceptors takes place almost exclusively via the TRP and TRPL channels.^25,28 Therefore, Ca²⁺ influx was measured during anoxia to identify the specific component of the response to anoxia, which arises directly from activation of these channels. Drosophila mutants, which are defective in proteins crucial for the phototransduction cascade, were used to localize the transduction stage that underlies the effects of anoxia. Using Ca²⁺-selective microelectrodes in WT flies, the well-described reduction in extracellular Ca²⁺ concentration ([Ca²⁺]_out) was measured during illumination (Fig. 3A, see also refs. 28,71). The reduction of [Ca²⁺]_out during illumination arises from Ca²⁺ influx into the photoreceptor cells due to openings of TRP and TRPL channels.²⁸ Application of anoxia indeed induced, after a delay, a reduction in [Ca²⁺]_out (Fig. 3A, bottom). This observation indicates that in response to anoxia the large depolarization phase and Ca²⁺ influx are due to openings of the light-sensitive channels. Fig. 3B shows that illumination of the blind mutant norpA^P24, in which light-activated PLC is missing,^7,72 did not elicit any response to light as monitored by either voltage or [Ca²⁺]_out changes as expected (Fig. 3B). Application of anoxia in the dark induced a voltage response, similar to that of WT with an initial small phase and a subsequent larger phase. The calcium signal, which accompanied the larger phase of the voltage change, was similar in WT and the mutant except for a faster onset in the mutant and an overshoot after anoxia was turned off (Fig. 3). The effects of anoxia on the ninaE^ora mutant, which is an opsin Rh1 null mutant,⁷³ and on the Gαq1 mutant, which has a highly reduced level of light-activated G-protein⁷⁴ were measured. The effects of anoxia on these mutants were similar to those observed in WT flies. The results thus show that anoxia affects a late stage of the phototransduction cascade downstream to PLC activation.

Figure 3

Anoxia activated the TRP and TRPL channels in both WT and PLC null mutant (norpA^P24) as monitored by Ca²⁺ influx. Extracellular voltage change (ERG, upper traces in both 3A and 3B) and potentiometric measurements with Ca²⁺-selective microelectrode (E (more...)

Additional demonstration that anoxia opens both TRP and TRPL channels in the dark was obtained by measuring Ca²⁺ influx in the trp mutant, in which TRP is missing, and in the trpl;trp double mutant, which lacks both channels.³³ The ERG response to light of the trp^P343 mutant revealed the typical decline towards baseline during illumination while the Ca²⁺ signal was transient and relatively small, as previously reported (Fig. 4A).²⁸ Application of anoxia to the trp^P343 mutant activated initially the slow and small phase of the voltage response (Fig. 4A). However, in contrast to WT and the other mutants mentioned above, a significantly smaller amplitudes of the second faster phase of the voltage and Ca²⁺ signal were observed in trp^P343 flies. The small Ca²⁺ signal in response to light and anoxia in the mutant reflects influx of Ca²⁺ into the photoreceptors via the TRPL channels.²⁸

Figure 4

Genetic elimination of TRP resulted in a reduced Ca²⁺ influx through the remaining TRPL channels in response to anoxia, while elimination of both TRP and TRPL completely abolished both excitation and Ca²⁺ influx. The same paradigm of Fig. 3 was repeated (more...)

An interesting characteristic of the response to anoxia of the trp^P343 mutant is the existence of a maintained component in both voltage and Ca²⁺ signals as long as anoxia was applied, as in WT (Fig. 4A). This maintained response to anoxia in the mutant is in sharp contrast to the transient nature of the response to continuous light.

To firmly establish that the Ca²⁺ influx in response to anoxia is due to activation of TRP and TRPL channels we measured [Ca²⁺]_out in response to anoxia in the double mutant trpl³⁰²;trp^P343. In this mutant the response to light is completely abolished (Fig. 4B).³³ Application of anoxia induced only the initial small voltage change with neither the subsequent larger voltage change nor any significant change in [Ca²⁺]_out during the initial 2 min of anoxia (Fig. 4).

The lack of virtually any Ca²⁺ influx in the trpl;trp double mutant in response to anoxia (Fig. 4) suggests that the mechanism which controls the opening of the TRP and TRPL channels is the target of anoxia.

Inhibition of Mitochondria Mimicked the Effects of Anoxia in Vivo

Fly photoreceptor cells are known to have a large number of mitochondria.⁷⁵ To investigate if the effects of anoxia in Drosophila retina are due to impaired function of the mitochondria, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were applied to the intact eye. DNP and CCCP are known uncouplers of the oxidative chain for ATP production in the mitochondria.⁷⁶ Application of DNP to the intact eyes of wild-type Drosophila induced a negative voltage change and abolished light excitation in a manner similar to the larger phase of the response to anoxia. The effects of DNP were partially reversible ∼20 min after the application. The effects of DNP on wild-type flies were accelerated when combined with either anoxia or illumination, thus suggesting that light stimulation and all forms of metabolic stress were additive. These experiments further suggest that mitochondria uncouplers mimicked the effects of anoxia in wild-type flies through effects on the TRP and TRPL channels. Whole-cell recordings in single photoreceptor cells added more conclusive evidence to the in vivo studies.

Mitochondrial Uncouplers and Depletion of ATP Activate the TRP and TRPL Channels in the Dark in Situ

Impairment of mitochondria function is expected to deplete ATP from the photoreceptor cells. To investigate more directly if depletion of ATP from the photoreceptor cells activates the TRP and TRPL channels, whole-cell patch clamp recordings in isolated ommatidia preparation was used.^22,23,25 When ATP and α-nicotinamide adenine dinucleotide (NAD) were omitted from the recording pipette of WT cells, a few light pulses of medium intensity (orange light) induced an inward current in the dark, after a delay of ∼100 s (Fig. 5A). Inclusion of ATP and NAD in the pipette prevented the induction of the inward dark current by repeated illumination, for at least 6 min. This observation suggests that although light pulses are known to reduce the ATP level in photoreceptor cells,⁷⁷ the supplement of exogenous ATP and NAD probably prevented depletion of ATP by illumination for at least 6 min. Application of 0.1 mM DNP to the bath (Fig. 5B) during recordings with pipettes without ATP and NAD induced the inward current in the dark in WT flies after a delay of only ∼20 s (Fig. 5B). When DNP (0.1 mM) was included in the pipette solution (without ATP and NAD) the inward current was induced in less than 30s from the onset of whole-cell recording.

Figure 5

Single-cell functional analysis by whole-cell recordings from newly eclosed flies, showing activation of the TRP and TRPL channels in WT and trp^P343 mutant, respectively, but not in the trpl;trp^P343 double mutant. Membrane currents are usually elicited (more...)

In either trp^P343 or in the double mutant trpl³⁰²;trp^P343, no dark current was elicited during prolonged recordings (<10 min) using pipettes without ATP and NAD. Application of 0.1 mM DNP either to the bath or into the pipette induced a noisy inward current of small amplitude in trp flies (Fig. 5D). In the double mutant trpl³⁰²;trp^P343 DNP (applied either to the bath or in the pipette) or CCCP, without ATP and NAD in the pipette had no effect (Fig. 5E), even after incubation of 16 min.

The inward current of WT cells, which was induced in the dark following illumination or DNP application in the absence of ATP and NAD, had all the characteristics of the TRP dependent current.^78,79 In WT cells in zero external Ca²⁺ the current showed inward and outward rectification (Fig. 5C) while the amplitude of inward current at negative holding potentials was greatly reduced in the presence of Ca²⁺ in the medium (Fig. 5C). La³⁺, a potent Ca²⁺ channel blocker that mimics the trp phenotype,^{25,29,31,32,78} completely blocked the TRP-dependent current (Fig. 5C), as previously described.⁷⁸ In the trp mutant, after application of DNP the resulting inward current had all the characteristics of the TRPL-dependent current of trp mutant flies. It had small amplitude, high noise (Fig. 5C) and nearly absence of inward rectification even at 0 Ca²⁺ level (see ref. 79).²⁰ The effects of DNP were additive with light or absence of ATP and NAD, thus illumination accelerated the onset of the effect of DNP while inclusion of ATP and NAD in the pipette increased the latency of TRP channels activation to 2–4 min.

In summary, the results show that elimination of ATP and NAD from the recording pipette, combined with conditions that deplete ATP from the mitochondria opened the TRP and TRPL channels in the dark by affecting a late stage of the transduction cascade.

This study thus demonstrates that activation of TRP and TRPL in the dark results from depletion of ATP. The depletion of ATP probably directly activates the channels and not earlier stages of the transduction cascade because of the following reasons: i) Anoxia activated the TRP and TRPL channels at a stage downstream to PLC because anoxia induced Ca²⁺ influx in the PLC null mutant, norpA^P24. ii) Activation of earlier stages results in production of unitary events (quantum bumps)^70,80 while depletion of ATP was shown to produce a smooth response with channel noise (see also ref. 78). iii) Application of anoxia to the null trp mutant in vivo and application of DNP in situ produced maintained openings of the TRPL channels.⁸¹

The results suggest that the mitochondria are the primary target of anoxia leading to a reduction in cellular ATP. Indeed, previous studies on the large fly Calliphora have demonstrated that application of N₂ rapidly and reversibly inhibited a specific transient green fluorescence that normally arises from functional mitochondria in the eye.⁸²

The observation that impaired mitochondia function leads to openings of TRP and TRPL channels has important implications on previous studies. In these studies activation of various TRP channels have been obtained by several means including oxidative stress⁸³ and application of polyunsaturated fatty acids (PuFAs).⁷⁰ The latter is of special interest because linoleic acid and several other long chain fatty acids have been shown to react as efficient uncouplers of mitochondria in a variety of cells.^84,85 The effect of DNP, therefore, suggest that PuFAs action on TRP and TRPL channels is indirect and result from their action as mitochondria uncouplers.⁸¹

The mechanism underlying activation of TRP and TRPL channels by depletion of ATP is not clear. However, the need to supply ATP in the dark at a high rate for proper function of the TRP and TRPL channels partially accounts for the well known, but unexplained, phenomenon of a high rate of oxygen consumption of insect retina in the dark.⁸⁶ The results, thus, suggest that a constitutive ATP dependent process operating at a late stage of the phototransduction cascade is required to keep the TRP and TRPL channels closed in the dark. This makes these channels extremely sensitive to anoxia and may lead to cell death under metabolic stress, not only in Drosophila but also in vertebrate cells, which express TRP channels.⁸³ The present study thus provides a novel target for metabolic stress with possible implications for brain damage since activation of TRP induces massive Ca²⁺ influx and TRP homologues are expressed in the brain.^36,39,52

Taken together, the link of TRP gating to the metabolic state of cells opens up a new avenue of research that may explain many of the seemingly contradictory reports on pharmacological properties and mechanism of activation of TRP channels.^36,39

Conclusion

The family of TRP channel proteins is unusually complex because of the large diversity of the structure and putative function of its members. Although the gating mechanism of any member of this large superfamily is unknown, there are indications that in the Drosophila light-activated channels and in some mammalian members, the gating of TRP is linked to the metabolic state of the cell. Although the function of the linkage between gating and metabolic stress is unknown, it put the cell in a great hazard because TRP channels are permeable to Ca²⁺. Accordingly, metabolic stress readily leads to toxic increase in cellular Ca²⁺ and cell death. Such a mechanism may underlie brain damage induced by ischemia, retinal degeneration in Drosophila, and potentially degeneration of retinal neurons expressing TRPs.

Acknowledgments

Experiments described in this paper were supported by the National Institutes of Health (EY 03529), the Israel Science Foundation (ISF), the German Israeli Foundation (GIF), the uS-Israel Binational Science Foundation (BSF), the Minerva Foundation and the Moscona Foundation.

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