Membrane lipid modulations by methyl-β-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating
2023; Elsevier BV; Volume: 300; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2023.105484
ISSN1083-351X
AutoresRita Gutorov, Ben Katz, Maximilian Peters, Baruch Minke,
Tópico(s)Bee Products Chemical Analysis
ResumoSterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein–coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MβCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MβCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MβCD suppressed a constitutively active TRP mutant–channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MβCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating. Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein–coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MβCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MβCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MβCD suppressed a constitutively active TRP mutant–channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MβCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating. Cyclodextrins are a family of cyclic oligosaccharides, consisting of glucose subunits arranged as macrocyclic rings. Chemically, the interior part of cyclodextrins is hydrophobic, while the exterior part is hydrophilic promoting formation of complexes with hydrophobic compounds. A common method for modulating sterol level in the plasma membrane is by incubation of cells with methyl-β-cyclodextrin (MβCD), a cyclic oligosaccharide consisting of a macrocyclic ring of seven glucose subunits joined by α-1,4 glycosidic bonds. MβCD has preferential binding toward sterols compared to phospholipids, allowing sequestration or enrichment of sterols of living cells membranes (1Ohtani Y. Irie T. Uekama K. Fukunaga K. Pitha J. Differential effects of alpha-, beta- and gamma-cyclodextrins on human erythrocytes.Eur. J. Biochem. 1989; 186: 17-22Crossref PubMed Scopus (564) Google Scholar). Sterol-saturated MβCD is efficient as sterol donor, enabling sterol enrichment by ∼30% to ∼3-fold (2Levitan I. Christian A.E. Tulenko T.N. Rothblat G.H. Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells.J. Gen. Physiol. 2000; 115: 405-416Crossref PubMed Scopus (178) Google Scholar, 3Christian A.E. Haynes M.P. Phillips M.C. 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In contrast, sterol sequestration has virtually no effect on phosphatidylinositol 4,5-bisphosphate (PIP2) clusters (PIP2 microdomains) in the inner-leaflet plasma membrane of cells, which are physically separated from sterol-containing lipid rafts (9van den Bogaart G. Meyenberg K. Risselada H.J. Amin H. Willig K.I. Hubrich B.E. et al.Membrane protein sequestering by ionic protein-lipid interactions.Nature. 2011; 479: 552-555Crossref PubMed Scopus (462) Google Scholar, 10Robinson C.V. Rohacs T. Hansen S.B. Tools for understanding nanoscale lipid regulation of ion channels trends.Biochem. Sci. 2019; 44: 795-806Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11Yuan Z. Hansen S.B. Cholesterol regulation of membrane proteins revealed by two-color super-resolution imaging.Membranes (Basel). 2023; 13: 250Crossref PubMed Scopus (5) Google Scholar). Drosophila phototransduction is a G-protein–coupled and phospholipase C (PLC)-mediated cascade, with transient receptor potential (TRP) and TRP-like (TRPL) as the transducer channels. Hydrolysis of PIP2 by PLC result in generation of diacylglycerol (DAG), reduction of PIP2 and generation of protons. All or some of these events are crucial for the physiological activation of TRP and TRPL channels and the generation of the light-induced current (LIC). However, the mechanism of channel gating is still under debate ((12Katz B. Minke B. Drosophila photoreceptors and signaling mechanisms.Front. Cell. Neurosci. 2009; 3: 2Crossref PubMed Scopus (105) Google Scholar, 13Hardie R.C. Juusola M. Phototransduction in Drosophila.Curr. Opin. Neurobiol. 2015; 34: 37-45Crossref PubMed Scopus (86) Google Scholar, 14Montell C. Drosophila visual transduction.Trends Neurosci. 2012; 35: 356-363Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 15Hardie R.C. Phototransduction in Drosophila melanogaster.J. Exp. Biol. 2001; 204: 3403-3409Crossref PubMed Google Scholar, 16Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. et al.Isolation of a putative phospholipase C gene of Drosophila , norpA , and its role in phototransduction.Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (569) Google Scholar, 17Delgado R. Muñoz Y. Peña-Cortés H. Giavalisco P. Bacigalupo J. Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.J. Neurosci. 2014; 34: 6679-6686Crossref PubMed Scopus (32) Google Scholar, 18Rhodes-Mordov E. Brandwine-Shemmer T. Zaguri R. Gutorov R. Peters M. Minke B. Diacylglycerol activates the Drosophila light sensitive channel TRPL expressed in HEK cells.Int. J. Mol. Sci. 2023; 24: 6289Crossref PubMed Scopus (1) Google Scholar), reviewed in (13Hardie R.C. Juusola M. Phototransduction in Drosophila.Curr. Opin. Neurobiol. 2015; 34: 37-45Crossref PubMed Scopus (86) Google Scholar, 19Katz B. Payne R. Minke B. TRP Channels in Vision. CRC Press, Taylor & Francis Group, Boca Raton, FL2017Crossref Google Scholar, 20Montell C. Visual transduction in Drosophila.Annu. Rev. Cell Dev. Biol. 1999; 15: 231-268Crossref PubMed Scopus (254) Google Scholar)). Mutations in proteins of the phosphoinositide cycle, which mediate conversion of the PLC product DAG back to PIP2 have been shown to induce light-dependent and light-independent photoreceptor degeneration and affect the TRP and TRPL channel activity (21Raghu P. Usher K. Jonas S. Chyb S. Polyanovsky A. Hardie R.C. Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA.Neuron. 2000; 26: 169-179Abstract Full Text Full Text PDF PubMed Google Scholar). Application of poly unsaturated fatty acids (PUFAs) robustly activated the TRP/TRPL channels in the dark in Drosophila photoreceptors (22Chyb S. Raghu P. Hardie R.C. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL.Nature. 1999; 397: 255-259Crossref PubMed Scopus (366) Google Scholar) in a PLC-independent manner (23Parnas M. Katz B. Minke B. Open channel block by Ca2+ underlies the voltage dependence of Drosophila TRPL channel.J. Gen. Physiol. 2007; 129: 17-28Crossref PubMed Scopus (32) Google Scholar). ATP depletion also activated the TRP/TRPL channels in the dark (24Agam K. von Campenhausen M. Levy S. Ben-Ami H.C. Cook B. Kirschfeld K. et al.Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.J. Neurosci. 2000; 20: 5748-5755Crossref PubMed Google Scholar), but in a PLC-dependent manner (25Hardie R.C. Martin F. Chyb S. Raghu P. Rescue of light responses in the Drosophila “null” phospholipase C mutant, norpAP24 by diacylglycerol kinase mutant, rdgA and by metabolic inhibition.J. Biol. Chem. 2003; 278: 18851-18858Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The activity of Drosophila TRPL channels expressed in tissue culture cells was shown to be suppressed by incubation with MβCD (26Peters M. Katz B. Lev S. Zaguri R. Gutorov R. Minke B. Depletion of membrane cholesterol suppresses Drosophila transient receptor potential-like (TRPL) channel activity.Curr. Top. Membr. 2017; 80: 233-254Crossref PubMed Scopus (11) Google Scholar). However, localizing the effect of sterol reduction on the phosphoinositide cascade that is natively expressed in these cells was not investigated. Unlike vertebrates, flies are unable to synthesize sterols (auxotroph) and receive this essential lipid compound from their yeasts diet in the form of ergosterol (27Eroglu C. Brugger B. Wieland F. Sinning I. Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10219-10224Crossref PubMed Scopus (90) Google Scholar). Dietary restriction of ergosterol intake of flies resulted in disruptive association of phototransduction signaling components with detergent-resistant membrane (DRM) lipid raft fractions (28Sanxaridis P.D. Cronin M.A. Rawat S.S. Waro G. Acharya U. Tsunoda S. Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors.Mol. Cell. Neurosci. 2007; 36: 36-46Crossref PubMed Scopus (23) Google Scholar). These signaling complexes included the scaffold protein inactivation-nο-afterpotential D (INAD), which binds major phototransduction components such as PLC and TRP. Importantly, these signaling proteins were found to be associated, in a light-dependent manner, with DRM lipid rafts domain. Hence, reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM lipid rafts fractions (28Sanxaridis P.D. Cronin M.A. Rawat S.S. Waro G. Acharya U. Tsunoda S. Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors.Mol. Cell. Neurosci. 2007; 36: 36-46Crossref PubMed Scopus (23) Google Scholar). However, the effects of ergosterol reduction by dietary restriction on the light response were not examined in this comprehensive biochemical study. In the present study we extended the previous reports, which reduced ergosterol levels in Drosophila photoreceptors by dietary manipulations, and examined, for the first time, the effect of ergosterol reduction by MβCD on PIP2 hydrolysis, when measured together with the physiological response to light. Accordingly, we used whole-cell voltage-clamp measurements from photoreceptor cells and simultaneously monitored PIP2 hydrolysis by translocation of fluorescently tagged lipid-binding Tubby protein domain, from the plasma membrane to the cytosol. These measurements revealed that incubation with MβCD virtually abolished the light response while having only little effect on the light activated PIP2 hydrolysis by PLC. Furthermore, MβCD suppressed a constitutively active TRP mutant–channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Together, MβCD uncoupled TRP/TRPL channel's gating from light-activated PLC and PIP2 hydrolysis, suggesting involvement of nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel's gating. Following the finding that application of MβCD suppresses the activity of the Drosophila TRPL channel expressed in tissue culture cells (26Peters M. Katz B. Lev S. Zaguri R. Gutorov R. Minke B. Depletion of membrane cholesterol suppresses Drosophila transient receptor potential-like (TRPL) channel activity.Curr. Top. Membr. 2017; 80: 233-254Crossref PubMed Scopus (11) Google Scholar), we thought of examining the effects of MβCD on the Drosophila TRP and TRPL channels in the native photoreceptor cells. First, we examined the effect of MβCD (10 mM), on the light response of the trpl302 null mutant (in which the LIC is composed only of TRP channels, Fig. 1, A–F) and trpP343 null mutant flies (in which the LIC is composed of only of TRPL channels, Fig. 1, G–K). Accordingly, the LIC in response to a train of brief intense orange light pulses, separated by dark intervals (60 s for trpl302 and 90 s for trpP343) were measured using the whole-cell voltage-clamp technique (trpl302 mutant, Fig. 1, A and B and trpP343 mutant Fig. 1, G–I), in the presence and absence of MβCD. The peak amplitude and latency (the time from light onset to the beginning of the response) of the LICs remained relatively constant under incubation with standard extracellular solution (SES) of both the trpl302 and trpP343 mutants (Fig. 1, B, E, and J, respectively), with only slight decrease in response amplitude and increase in response latency after ∼9 min (#8 light pulse for trpl302, #5 light pulse for trpP343, Fig. 1, F and K, respectively). Interestingly, incubation of the photoreceptor cells of the trpl302 with MβCD significantly reduced the amplitude and increased the latency of the LIC (Fig. 1, E and F), while incubation of the photoreceptor cells of the trpP343 with MβCD significantly reduced the light response amplitude but increased the response latency only to a small extent (Fig. 1, J and K). In summary, incubation of photoreceptor cells from trpl302 (expressing only the TRP channel) and trpP343 flies (expressing only the TRPL channel) with MβCD decreased the amplitude and slowed the kinetics of the LIC. Drosophila photoreceptor cells have reached the ultimate sensitivity to light, by responding to absorption of single photons with discrete voltage (or current) change called quantum bump (29Yeandle S. Spiegler J.B. Light-evoked and spontaneous discrete waves in the ventral nerve photoreceptor of Limulus.J. Gen. Physiol. 1973; 61: 552-571Crossref PubMed Google Scholar, 30Wu C.F. Pak W.L. Quantal basis of photoreceptor spectral sensitivity of Drosophila melanogaster.J. Gen. Physiol. 1975; 66: 149-168Crossref PubMed Google Scholar). In WT flies, a quantum bump is the result of synchronized activation of several TRP and TRPL light-sensitive channels in a single microvillus (31Song Z. Postma M. Billings S.A. Coca D. Hardie R.C. Juusola M. Stochastic, adaptive sampling of information by microvilli in fly photoreceptors.Curr. Biol. 2012; 22: 1371-1380Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Quantum bump of WT and the trpl302 mutant flies (expressing only the TRP channel) display large amplitude (∼8–14 pA) under standard condition (32Katz B. Gutorov R. Rhodes-Mordov E. Hardie R.C. Minke B. Electrophysiological method for whole-cell voltage clamp recordings from Drosophila photoreceptors.J. Vis. Exp. 2017; https://doi.org/10.3791/55627Crossref PubMed Scopus (5) Google Scholar, 33Henderson S.R. Reuss H. Hardie R.C. Single photon responses in Drosophila photoreceptors and their regulation by Ca 2+.J. Physiol. 2000; 524 Pt 1: 179-194Crossref PubMed Scopus (119) Google Scholar) that can be readily measured and analyzed. However, quantum bumps of trpP343 mutant flies (expressing only the TRPL channel) are small in amplitude (∼3 pA), making measurement and analysis extremely challenging and therefore bump analysis was performed only on trpl302 mutant photoreceptors. Since the macroscopic LIC constitutes a summation of quantum bumps (34Barash S. Minke B. Is the receptor potential of fly photoreceptors a summation of single-photon responses?.Theor. Biol. 1994; 3: 229-263Google Scholar, 35Wong F. Knight B.W. Dodge F.A. Adapting bump model for ventral photoreceptors of Limulus.J. Gen. Physiol. 1982; 79: 1089-1113Crossref PubMed Google Scholar), changes in bumps amplitude, frequency, waveform, or latency distribution affect the macroscopic light response. To analyze the effect of MβCD on bump parameters, we recorded single photon responses under dim light illumination from the trpl302 mutant flies (expressing TRP channels) in the presence and absence of MβCD. Bumps recordings and analysis under standard condition from photoreceptors of trpl302 mutant flies revealed that the bump amplitude was relatively stable (∼13 pA, see (36Katz B. Minke B. Phospholipase C-mediated suppression of dark noise enables single-photon detection in Drosophila photoreceptors.J. Neurosci. 2012; 32: 2722-2733Crossref PubMed Scopus (24) Google Scholar)) during 15 min of recording (Fig. 2C, control). Bump frequency under standard condition started at ∼2 bumps/s and decreased by ∼25% reaching ∼1.5 bumps/s after 15 min of recording under constant continuous dim light (Fig. 2D, MβCD, control (33Henderson S.R. Reuss H. Hardie R.C. Single photon responses in Drosophila photoreceptors and their regulation by Ca 2+.J. Physiol. 2000; 524 Pt 1: 179-194Crossref PubMed Scopus (119) Google Scholar)). Incubation of the photoreceptors with MβCD caused a time-dependent decrease in bump frequency reaching a reduction of ∼85% after ∼10 min (Fig. 2, B and D). In contrast, incubation of the photoreceptors with MβCD had relatively small effect on the bump's amplitude, which began showing reduced amplitudes only after ∼7 min and reaching a ∼50% reduction after ∼10 min (Fig. 2, B and C, MβCD). At the 10.5 to 12 min time point of incubation with 10 mM MβCD, the mean bump amplitude was reduced to ∼7 pA and mean bump frequency to ∼0.27 bumps per second. To emphasize the differential effect of MβCD on bump amplitude relative to bump frequency, a histogram is shown in Figure 2E presenting the reduction in the normalized mean bump amplitude (blue columns) and mean bump frequency (orange columns) relative to their control, (% difference). The histogram shows a significant larger reduction in bump frequency as than the reduction in bump amplitude following application of MβCD (Fig. 2E, see the implications in the Discussion). Previous studies have shown that it is possible to bypass the light activation of key signal transduction components of the phototransduction cascade and activate pharmacologically the TRP and TRPL channels in the dark in vivo. This method can assist in identifying molecular components of the phototransduction cascade, which are sensitive to pharmacological agents. One such way is to induce metabolic inhibition by anoxia in vivo (24Agam K. von Campenhausen M. Levy S. Ben-Ami H.C. Cook B. Kirschfeld K. et al.Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.J. Neurosci. 2000; 20: 5748-5755Crossref PubMed Google Scholar) or by depleting ATP from the photoreceptor cell ex vivo, using specific mitochondrial uncouplers such as carbonyl cyanide m-chlorophenyl hydrazine (CCCP, (24Agam K. von Campenhausen M. Levy S. Ben-Ami H.C. Cook B. Kirschfeld K. et al.Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.J. Neurosci. 2000; 20: 5748-5755Crossref PubMed Google Scholar)). Under conditions of ATP depletion, dark activation of the TRP/TRPL channels is not manifested by synchronous channel activation like channel activation by the absorbed photons (quantum bumps) but rather by continuous noisy slow inward current called rundown current (RDC), which is composed of channel noise that arises directly from non-synchronous activation of the TRP (Fig. 3A top, Fig. 3C control, left) and TRPL channels (Fig. 3B, top, Fig. 3C control, right (24Agam K. von Campenhausen M. Levy S. Ben-Ami H.C. Cook B. Kirschfeld K. et al.Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.J. Neurosci. 2000; 20: 5748-5755Crossref PubMed Google Scholar, 37Hardie R.C. Minke B. Spontaneous activation of light-sensitive channels in Drosophila photoreceptors.J. Gen. Physiol. 1994; 103: 389-407Crossref PubMed Google Scholar)). A suggested explanation for the ATP depletion–mediated dark channel activation came from studies of flies mutated in the retinal degeneration A (rdgA) gene encoding for DAG kinase (38Masai I. Okazaki A. Hosoya T. Hotta Y. Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11157-11161Crossref PubMed Scopus (141) Google Scholar). The rdgA mutant fly shows light-independent retinal degeneration (39Benzer S. Genetic dissection of behavior.Sci. Am. 1973; 229: 24-37Crossref PubMed Google Scholar) and dark activity of the TRP/TRPL channels (21Raghu P. Usher K. Jonas S. Chyb S. Polyanovsky A. Hardie R.C. Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA.Neuron. 2000; 26: 169-179Abstract Full Text Full Text PDF PubMed Google Scholar) similar to that observed, following TRP and TRPL channel activation by mitochondrial uncouplers (24Agam K. von Campenhausen M. Levy S. Ben-Ami H.C. Cook B. Kirschfeld K. et al.Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.J. Neurosci. 2000; 20: 5748-5755Crossref PubMed Google Scholar, 40Agam K. Frechter S. Minke B. Activation of the Drosophila TRP and TRPL channels requires both Ca 2+ and protein dephosphorylation.Cell Calcium. 2004; 35: 87-105Crossref PubMed Scopus (26) Google Scholar). Therefore, it was suggested that cellular ATP depletion promotes TRP and TRPL channel opening as a result of DAG accumulation caused by the inhibition of DAG kinase, either directly by the rdgA mutation or indirectly by ATP depletion. The presumed accumulation of DAG in the dark in the rdgA mutants or in ATP-depleted photoreceptors was suggested to arise from a small basal (leak) activity of PLC (41Hardie R.C. Regulation of trp channels via lipid second messengers.Annu. Rev. Physiol. 2003; 65: 735-759Crossref PubMed Scopus (201) Google Scholar). Hence, activation of the TRP and TRPL channels by ATP depletion does not involve signaling proteins upstream of PLC. In order to identify the molecular component in the signal transduction cascade, which is affected by incubation with MβCD, the response of trpl302 and trpP343 mutant photoreceptors to metabolic inhibition (using CCCP) in the dark was measured in the absence and presence of MβCD. MβCD strongly suppressed both the TRP- and TRPL-dependent currents (TRP: Fig. 3A, bottom, Fig. 3C, red, left; TRPL: Fig. 3B, bottom, Fig. 3C, red, right) that were induced by CCCP application in the dark. Since the CCCP-induced TRP and TRPL currents depend on a leak of PLC activity in the dark (42Hardie R.C. Gu Y. Martin F. Sweeney S.T. Raghu P. In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1).J. Biol. Chem. 2004; 279: 47773-47782Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the results suggest that at least some of the effect of MβCD is either at the PLC level or downstream of PLC activation (e.g., at the level of the light activated channels). The activation of PLC by light is a crucial step in the physiological activation of TRP and TRPL channels (12Katz B. Minke B. Drosophila photoreceptors and signaling mechanisms.Front. Cell. Neurosci. 2009; 3: 2Crossref PubMed Scopus (105) Google Scholar, 13Hardie R.C. Juusola M. Phototransduction in Drosophila.Curr. Opin. 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Shan W.S. et al.G-protein signaling through tubby proteins.Science. 2001; 292: 2041-2050Crossref PubMed Scopus (312) Google Scholar), while a point mutation of the same construct, TbR332H, reports cellular PIP2 changes independently of inositol-tris-phosphate generation (44Quinn K.V. Behe P. Tinker A. Monitoring changes in membrane phosphatidylinositol 4,5-bisphosphate in living cells using a domain from the transcription factor tubby.J. Physiol. 2008; 586: 2855-2871Crossref PubMed Scopus (72) Google Scholar) and thus fits best our experiments. Upon PIP2 hydrolysis by intense blue light activation of PLC, the fluorescent TbR332H-YFP probe translocate from the plasma membrane to the cytosol of the photoreceptor cells enabling an estimated measure of PLC hydrolyzing activity (45Liu C.H. Bollepalli M.K. Long S.V. Asteriti S. Tan J. Brill J.A. et al.Genetic dissection of the phosphoinositide cycle in Drosophila photoreceptors.J. Cell Sci. 2018; 131jcs214478Google Scholar, 46Hardie R.C. 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