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Articles by Baruch Minke in JoVE
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Электрофизиологический метод записи цельного клеточного напряжения
Ben Katz*1, Rita Gutorov*1, Elisheva Rhodes-Mordov1, Roger C. Hardie2, Baruch Minke1
1Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Hebrew University, 2Department of Physiology, Development and Neuroscience, University of Cambridge
Запись целых клеток из фоторецепторов Drosophila melanogaster позволяет измерять спонтанные темные удары, квантовые удары, макроскопические ответы на свет и отношения напряжения и напряжения при различных условиях. В сочетании с инструментами генетической манипуляции D. melanogaster этот метод позволяет изучать вездесущий сигнальный путь inositol-lipid и его целевой канал TRP.
Other articles by Baruch Minke on PubMed
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TRP Channel Proteins and Signal Transduction
Physiological Reviews.
Apr, 2002 |
Pubmed ID: 11917094 TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.
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Light-regulated Subcellular Translocation of Drosophila TRPL Channels Induces Long-term Adaptation and Modifies the Light-induced Current
Neuron.
Mar, 2002 |
Pubmed ID: 11931743 Drosophila phototransduction results in the opening of two classes of cation channels, composed of the channel subunits transient receptor potential (TRP), TRP-like (TRPL), and TRPgamma. Here, we report that one of these subunits, TRPL, is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL, characteristic of dark-raised flies, is functionally manifested in the properties of the light-induced current. These flies are more sensitive than flies with no or reduced TRPL level to dim background lights, and they respond to a wider range of light intensities, which fit them to function better in darkness or dim background illumination. Thus, TRPL translocation represents a novel mechanism to fine tune visual responses.
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Excess of Gbetae over Gqalphae in Vivo Prevents Dark, Spontaneous Activity of Drosophila Photoreceptors
The Journal of Cell Biology.
Nov, 2005 |
Pubmed ID: 16260498 Drosophila melanogaster photoreceptor cells are capable of detecting single photons. This utmost sensitivity is critically dependent on the maintenance of an exceedingly low, dark, spontaneous activity of photoreceptor cells. However, the underlying mechanisms of this hallmark of phototransduction are not fully understood. An analysis of the Drosophila visual heterotrimeric (alphabetagamma) Gq protein revealed that wild-type Drosophila flies have about a twofold excess of Gbeta over Galpha subunits of the visual Gq protein. Studies of Gbetae mutants in which the excess of Gbeta was genetically eliminated showed dramatic dark, spontaneous activity of the photoreceptor cells, whereas concurrent genetic reduction of the Galpha subunit, which restored the excess of Gbeta, abolished this effect. These results indicate that an excess of Gbeta over Galpha is a strategy used in vivo for the suppression of spontaneous activity, thereby yielding a high signal to noise ratio, which is characteristic of the photoreceptor light response. This mechanism could be relevant to the regulation of G protein signaling in general.
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Light-regulated Translocation of Signaling Proteins in Drosophila Photoreceptors
Journal of Physiology, Paris.
Mar-May, 2006 |
Pubmed ID: 16458490 Illumination of Drosophila photoreceptor cells induces multi-facet responses, which include generation of the photoreceptor potential, screening pigment migration and translocation of signaling proteins which is the focus of recent extensive research. Translocation of three signaling molecules is covered in this review: (1) Light-dependent translocation of arrestin from the cytosol to the signaling membrane, the rhabdomere, determines the lifetime of activated rhodopsin. Arrestin translocates in PIP3 and NINAC myosin III dependent manner, and specific mutations which disrupt the interaction between arrestin and PIP3 or NINAC also impair the light-dependent translocation of arrestin and the termination of the response to light. (2) Activation of Drosophila visual G protein, DGq, causes a massive and reversible, translocation of the alpha subunit from the signaling membrane to the cytosol, accompanied by activity-dependent architectural changes. Analysis of the translocation and the recovery kinetics of DGq(alpha) in wild-type flies and specific visual mutants indicated that DGq(alpha) is necessary but not sufficient for the architectural changes. (3) The TRP-like (TRPL) but not TRP channels translocate in a light-dependent manner between the rhabdomere and the cell body. As a physiological consequence of this light-dependent modulation of the TRP/TRPL ratio, the photoreceptors of dark-adapted flies operate at a wider dynamic range, which allows the photoreceptors enriched with TRPL to function better in darkness and dim background illumination. Altogether, signal-dependent movement of signaling proteins plays a major role in the maintenance and function of photoreceptor cells.
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Insights on TRP Channels from in Vivo Studies in Drosophila
Annual Review of Physiology.
2006 |
Pubmed ID: 16460287 Transient receptor potential (TRP) channels mediate responses in a large variety of signaling mechanisms. Most studies on mammalian TRP channels rely on heterologous expression, but their relevance to in vivo tissues is not entirely clear. In contrast, Drosophila TRP and TRP-like (TRPL) channels allow direct analyses of in vivo function. In Drosophila photoreceptors, activation of TRP and TRPL is mediated via the phosphoinositide cascade, with both Ca2+ and diacylglycerol (DAG) essential for generating the light response. In tissue culture cells, TRPL channels are constitutively active, and lipid second messengers greatly facilitate this activity. Inhibition of phospholipase C (PLC) completely blocks lipid activation of TRPL, suggesting that lipid activation is mediated via PLC. In vivo studies in mutant Drosophila also reveal an acute requirement for lipid-producing enzyme, which may regulate PLC activity. Thus, PLC and its downstream second messengers, Ca2+ and DAG, constitute critical mediators of TRP/TRPL gating in vivo.
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Subcellular Translocation of the EGFP-tagged TRPL Channel in Drosophila Photoreceptors Requires Activation of the Phototransduction Cascade
Journal of Cell Science.
Jun, 2006 |
Pubmed ID: 16735439 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. We have recently found that 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. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could 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, Galphaq, phospholipase Cbeta and the TRP ion channel, or in proteins required for TRP function. Our 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, we 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+.
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TRP Channels and Ca2+ Signaling
Cell Calcium.
Sep, 2006 |
Pubmed ID: 16806461 There is a rapidly growing interest in the family of transient receptor potential (TRP) channels because TRP channels are not only important for many sensory systems, but they are crucial components of the function of neurons, epithelial, blood and smooth muscle cells. These facts make TRP channels important targets for treatment of diseases arising from the malfunction of these channels in the above cells and for treatment of inflammatory pain. TRP channels are also important for a growing number of genetic diseases arising from mutations in various types of TRP channels. The Minerva-Gentner Symposium on TRP channels and Ca(2+) signaling, which took place in Eilat, Israel (February 24-28, 2006) has clearly demonstrated that the study of TRP channels is a newly emerging field of biomedicine with prime importance. In the Eilat symposium, investigators who have contributed seminal publications and insight into the TRP field presented their most recent, and in many cases still unpublished, studies. The excellent presentations and excitement generated by them demonstrated that much progress has been achieved. Nevertheless, it was also evident that the field of TRP channels is still in its infancy in comparison to other fields of ion channels, and even the fundamental knowledge of the gating mechanism of TRP channels is still unsolved. The beautiful location of the symposium, together with informal intensive discussions among the participants, contributed to the success of this meeting.
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Translocation of Gq Alpha Mediates Long-term Adaptation in Drosophila Photoreceptors
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience.
May, 2007 |
Pubmed ID: 17522302 Light adaptation is a process that enables photoreceptor cells to operate over a wide range of light intensities without saturation. In invertebrate photoreceptors, fast adaptation is mediated by a Ca2+-dependent negative-feedback mechanism, which mainly affects the terminal steps of the cascade. Therefore, the response to each photon is smaller as light intensity increases, accommodating both high sensitivity and a vast dynamic range. Here, we describe a novel type of adaptation, which is mediated by one of the first steps in the phototransduction cascade affecting the sensitivity to absorbed photons. Long exposure to light resulted in dramatic reduction in the probability of each absorbed photon to elicit a response, whereas the size and shape of each single photon response did not change. To dissect the molecular mechanism underlying this form of adaptation we used a series of Drosophila mutants. Genetic dissection showed a pivotal role for light-induced translocation of Gq alpha between the signaling membrane and the cytosol. Biochemical studies revealed that the sensitivity to light depends on membrane Gq alpha concentration, which was modulated either by light or by mutations that impaired its targeting to the membrane. We conclude that long-term adaptation is mediated by the movement of Gq alpha from the signaling membrane to the cytosol, thereby reducing the probability of each photon to elicit a response. The slow time scale of this adaptation fits well with day/night light intensity changes, because there is no need to maintain single photon sensitivity during daytime.
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Carvacrol is a Novel Inhibitor of Drosophila TRPL and Mammalian TRPM7 Channels
Cell Calcium.
Mar, 2009 |
Pubmed ID: 19135721 Transient receptor potential (TRP) channels are essential components of biological sensors that detect changes in the environment in response to a myriad of stimuli. A major difficulty in the study of TRP channels is the lack of pharmacological agents that modulate most members of the TRP superfamily. Notable exceptions are the thermoTRPs, which respond to either cold or hot temperatures and are modulated by a relatively large number of chemical agents. In the present study we demonstrate by patch clamp whole cell recordings from Schneider 2 and Drosophila photoreceptor cells that carvacrol, a known activator of the thermoTRPs, TRPV3 and TRPA1 is an inhibitor of the Drosophila TRPL channels, which belongs to the TRPC subfamily. We also show that additional activators of TRPV3, thymol, eugenol, cinnamaldehyde and menthol are all inhibitors of the TRPL channel. Furthermore, carvacrol also inhibits the mammalian TRPM7 heterologously expressed in HEK cells and ectopically expressed in a primary culture of CA3-CA1 hippocampal brain neurons. This study, thus, identifies a novel inhibitor of TRPC and TRPM channels. Our finding that the activity of the non-thermoTRPs, TRPL and TRPM7 channels is modulated by the same compound as thermoTRPs, suggests that common mechanisms of channel modulation characterize TRP channels.
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Linoleic Acid Inhibits TRP Channels with Intrinsic Voltage Sensitivity: Implications on the Mechanism of Linoleic Acid Action
Channels (Austin, Tex.).
May-Jun, 2009 |
Pubmed ID: 19535910 Open channel block (OCB) is a process by which ions bind to the inside of a channel pore and block the flow of ions through that channel. Repulsion of the blocking ions by membrane depolarization is a known mechanism for open channel block removal. For the N-methyl-D-aspartate (NMDA) channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of Transient Receptor Potential (TRP) channels, including the Drosophila TRP and TRP-Like (TRPL) channels, also exhibit open channel block. For the Drosophila TRP and TRPL channels, removal of open channel block is necessary for the production of the physiological response to light. Recently, we have shown that lipids such as polyunsaturated fatty acids (PUFAs), represented by linoleic acid (LA), alleviate OCB under physiological conditions, from the Drosophila TRP and TRPL channels and from the mammalian NMDA channel. Here we show that OCB removal by LA is not confined to the Drosophila TRPs but also applies to mammalian TRPs such as the heat activated TRPV3 channel. TRPV3 shows OCB alleviation by LA, although it shares little amino acid sequence homology with the Drosophila TRPs. Strikingly, LA inhibits the heat-activated TRPV1 and the cold temperature-activated TRPM8 channels, which are intrinsic voltage sensitive channels and do not show OCB. Together, our findings further support the notion that lipids do not act as second messengers by direct binding to a specific site of the channels but rather act indirectly by affecting the channel-plasma membrane interface.
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Constitutive Activity of the Human TRPML2 Channel Induces Cell Degeneration
The Journal of Biological Chemistry.
Jan, 2010 |
Pubmed ID: 19940139 The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca(2+)-permeable and inhibited by low extracytosolic pH but not Ca(2+) regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca(2+) overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation.
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Cellular Functions of Transient Receptor Potential Channels
The International Journal of Biochemistry & Cell Biology.
Sep, 2010 |
Pubmed ID: 20399884 Transient Receptor Potential channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by increasing cellular Ca(2+). In this review we focus on the roles of these channels in: (i) cell death (ii) proliferation and differentiation and (iii) transmitter release. Cell death: Ca(2+) influx participates in apoptotic and necrotic cell death. The Ca(2+) permeability and high sensitivity of part of these channels to oxidative/metabolic stress make them important participants in cell death. Several examples are given. Transient Receptor Potential Melastatin 2 is activated by H(2)O(2), inducing cell death through an increase in cellular Ca(2+) and activation of Poly ADP-Ribose Polymerase. Exposure of cultured cortical neurons to oxygen-glucose deprivation, in vitro, causes cell death via cation influx, mediated by Transient Receptor Potential Melastatin 7. Metabolic stress constitutively activates the Ca(2+) permeable Transient Receptor Potential channels of Drosophila photoreceptor in the dark, potentially leading to retinal degeneration. Similar sensitivity to metabolic stress characterizes several mammalian Transient Receptor Potential Canonical channels. Proliferation and differentiation: The rise in cytosolic Ca(2+) induces cell growth, differentiation and proliferation via activation of several transcription factors. Activating a variety of store operated and Transient Receptor Potential channels cause a rise in cytosolic Ca(2+), making these channels components involved in proliferation and differentiation. Transmitter release: Transient Receptor Potential Melastatin 7 channels reside in synaptic vesicles and regulate neurotransmitter release by a mechanism that is not entirely clear. All the above features of Transient Receptor Potential channels make them crucial components in important, sometimes conflicting, cellular processes that still need to be explored.
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Heteromultimeric TRPML Channel Assemblies Play a Crucial Role in the Regulation of Cell Viability Models and Starvation-induced Autophagy
Journal of Cell Science.
Sep, 2010 |
Pubmed ID: 20736310 The mucolipin (TRPML) subfamily of transient receptor potential (TRP) cation channels consists of three members that play various roles in the regulation of membrane and protein sorting along endo-lysosomal pathways. Loss-of-function mutations in TRPML1 cause the neurodegenerative lysosomal storage disorder, mucolipidosis type IV (MLIV), whereas a gain-of-function mutation in TRPML3 is principally implicated in the hearing-impaired and abnormally pigmented varitint-waddler mouse. Currently, TRPML2 is not implicated in any pathological disorder, but we have recently shown that it is a functional cation channel that physically interacts with TRPML1 and TRPML3 to potentially regulate lysosomal integrity. Here, we show that mutant TRPMLs heteromultimerize with other mutant and wild-type TRPMLs to regulate cell viability and starvation-induced autophagy, a process that mediates macromolecular and organellar turnover under cell starvation conditions. Heteromultimerization of dominant-negative TRPMLs with constitutively active TRPMLs rescues cells from the cytotoxic effects of TRPML constitutive activity. Moreover, dominant-negative TRPML1 channels, including a mutant channel directly implicated in MLIV pathology, also inhibit starvation-induced autophagy by interacting with and affecting native TRPML channel function. Collectively, our results indicate that heteromultimerization of TRPML channels plays a role in various TRPML-regulated mechanisms.
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The History of the Drosophila TRP Channel: the Birth of a New Channel Superfamily
Journal of Neurogenetics.
Dec, 2010 |
Pubmed ID: 21067449 Transient receptor potential (TRP) channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by changing membrane voltage and increasing cellular Ca(2+). This review outlines in detail the history of the founding member of the TRP family, the Drosophila TRP channel. The field began with a spontaneous mutation in the trp gene that led to a blind mutant during prolonged intense light. It was this mutant that allowed for the discovery of the first TRP channels. A combination of electrophysiological, biochemical, Ca(2+) measurements, and genetic studies in flies and in other invertebrates pointed to TRP as a novel phosphoinositide-regulated and Ca(2+)-permeable channel. The cloning and sequencing of the trp gene provided its molecular identity. These seminal findings led to the isolation of the first mammalian homologues of the Drosophila TRP channels. We now know that TRP channel proteins are conserved through evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN. A great deal is known today about participation of TRP channels in many biological processes, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, cardiovascular regulation, smooth muscle tone, pressure regulation, Ca(2+) and Mg(2+) homeostasis, and lysosomal function. The native Drosophila photoreceptor cells, where the founding member of the TRP channels superfamily was found, is still a useful preparation to study basic features of this remarkable channel.
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Signal-dependent Hydrolysis of Phosphatidylinositol 4,5-bisphosphate Without Activation of Phospholipase C: Implications on Gating of Drosophila TRPL (transient Receptor Potential-like) Channel
The Journal of Biological Chemistry.
Jan, 2012 |
Pubmed ID: 22065576 In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.
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The History of the Prolonged Depolarizing Afterpotential (PDA) and Its Role in Genetic Dissection of Drosophila Phototransduction
Journal of Neurogenetics.
Jun, 2012 |
Pubmed ID: 22428622 In invertebrate photoreceptors, the photopigment exhibits a long-lived and physiologically active photoproduct, called metarhodopsin (M). The long life of invertebrate M implies that under physiological conditions, M and the original pigment state rhodopsin, R, are in photoequilibrium. In many invertebrates, the absorption spectra of R and M states are different, allowing large photopigment conversion between R and M states. These net pigment molecules conversions between R and M are the basis of the prolonged depolarizing afterpotential (PDA) phenomenology, which is the main subject of this review. A large net conversion of R to M disrupts phototransduction termination at the photopigment level, which in turn results in sustained excitation long after the light is turned off. Throughout this period, the photoreceptors are partially desensitized and are insensitive (or less sensitive) to subsequent test lights. In Drosophila, the PDA tests the maximal capacity of the photoreceptor cell to maintain excitation for an extended period and is strictly dependent on the presence of high concentrations of rhodopsin and the transient receptor potential (TRP) channels. Therefore, it detects even minor defects in rhodopsin or TRP biogenesis and easily scores deficient replenishment of phototransduction components, which results in temporary desensitization of the phototransduction process. Indeed, the introduction and use of PDA to screen for phototransduction-defective Drosophila mutants by Pak and colleagues yielded a plethora of new and most interesting visual mutants. Remarkably, to this day, the PDA mutants that Pak and his colleagues isolated are the main source of mutants for analysis of the Drosophila visual system.
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Carvacrol Together with TRPC1 Elimination Improve Functional Recovery After Traumatic Brain Injury in Mice
Journal of Neurotrauma.
Dec, 2012 |
Pubmed ID: 22994850 Death of Central Nervous System (CNS) neurons following traumatic brain injury (TBI) is a complex process arising from a combination of factors, many of which are still unknown. It has been found that inhibition of transient receptor potential (TRP) channels constitutes an effective strategy for preventing death of CNS neurons following TBI. TRP channels are classified into seven related subfamilies, most of which are Ca(2+) permeable and involved in many cellular functions, including neuronal cell death. We hypothesized that TRP channels of the TRPC subfamily may be involved in post-TBI pathophysiology and that the compound 5-isopropyl-2-methylphenol (carvacrol), by inhibition of TRP channels, may exert neuroprotective effect after TBI. To test these suppositions, carvacrol was given to mice after TBI and its effect on their functional recovery was followed for several weeks. Our results show that neurological recovery after TBI was significantly enhanced by application of carvacrol. To better define the type of the specific channel involved, the effect of carvacrol on the extent and speed of recovery after TBI was compared among mice lacking TRPC1, TRPC3, or TRPC5, relative to wild type controls. We found that neurological recovery after TBI was significantly enhanced by combining carvacrol with TRPC1 elimination, but not by the absence of TRPC3 or TRPC5, showing a synergistic effect between carvacrol application and TRPC1 elimination. We conclude that TRPC1-sensitive mechanisms are involved in TBI pathology, and that inhibition of this channel by carvacrol enhances recovery and should be considered for further studies in animal models and humans.
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Compartmentalization and Ca2+ Buffering Are Essential for Prevention of Light-induced Retinal Degeneration
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience.
Oct, 2012 |
Pubmed ID: 23077055 Fly photoreceptors are polarized cells, each of which has an extended interface between its cell body and the light-signaling compartment, the rhabdomere. Upon intense illumination, rhabdomeric calcium concentration reaches millimolar levels that would be toxic if Ca(2+) diffusion between the rhabdomere and cell body was not robustly attenuated. Yet, it is not clear how such effective attenuation is obtained. Here we show that Ca(2+) homeostasis in the photoreceptor cell relies on the protein calphotin. This unique protein functions as an immobile Ca(2+) buffer localized along the base of the rhabdomere, separating the signaling compartment from the cell body. Generation and analyses of transgenic Drosophila strains, in which calphotin-expression levels were reduced in a graded manner, showed that moderately reduced calphotin expression impaired Ca(2+) homeostasis while calphotin elimination resulted in severe light-dependent photoreceptor degeneration. Electron microscopy, electrophysiology, and optical methods revealed that the degeneration was rescued by prevention of Ca(2+) overload via overexpression of CalX, the Na(+)-Ca(2+) exchanger. In addition, Ca(2+)-imaging experiments showed that reduced calphotin levels resulted in abnormally fast kinetics of Ca(2+) elevation in photoreceptor cells. Together, the data suggest that calphotin functions as a Ca(2+) buffer; a possibility that we directly demonstrate by expressing calphotin in a heterologous expression system. We propose that calphotin-mediated compartmentalization and Ca(2+) buffering constitute an effective strategy to protect cells from Ca(2+) overload and light-induced degeneration.
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Drosophila TRP and TRPL Are Assembled As Homomultimeric Channels in Vivo
Journal of Cell Science.
Jul, 2013 |
Pubmed ID: 23687378 Family members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.
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A New Genetic Model for Calcium Induced Autophagy and ER-stress in Drosophila Photoreceptor Cells
Channels (Austin, Tex.).
2015 |
Pubmed ID: 25664921 Cytoplasmic Ca2+ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca2+ buffer, which participates in Ca2+ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca2+ influx into photoreceptor cells, resulted in severe Ca2+-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca2+ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca2+, ER-stress and autophagy in human disorders and diseases.
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Functional Cooperation Between the IP3 Receptor and Phospholipase C Secures the High Sensitivity to Light of Drosophila Photoreceptors in Vivo
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience.
Feb, 2015 |
Pubmed ID: 25673847 Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. We have recently shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLCβ activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. We show here that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCβ catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, we show that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP3R found here may represent a common feature of the inositol-lipid signaling.
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Ectopic Expression of Mouse Melanopsin in Drosophila Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation
The Journal of Biological Chemistry.
Mar, 2017 |
Pubmed ID: 28119450 The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was ∼40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin.
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The Phosphorylation State of the Drosophila TRP Channel Modulates the Frequency Response to Oscillating Light In Vivo
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience.
Apr, 2017 |
Pubmed ID: 28314815 Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca(2+)-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trp(S936A) ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trp(S936D) ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions.SIGNIFICANCE STATEMENTDrosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤∼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca(2+)-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions.
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