Other articles by Roger C. Hardie on PubMed

• The Target of Drosophila Photoreceptor Synaptic Transmission is a Histamine-gated Chloride Channel Encoded by Ort (hclA) The Journal of Biological Chemistry. Nov, 2002  |   Pubmed ID: 12196539 By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), we identified ort as a candidate gene encoding a histamine receptor subunit on L1/L2. We provide evidence that the ort gene corresponds to CG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using cloned hclA cDNA and by showing that several ort mutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions in hclA transcript levels. Other workers have shown that hclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. We show unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclA and ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo.
• Regulation of TRP Channels Via Lipid Second Messengers Annual Review of Physiology. 2003  |   Pubmed ID: 12560473 In Drosophila photoreceptors, the light-sensitive current is mediated downstream of phospholipase C by TRP (transient receptor potential) channels. Recent evidence suggests that Drosophila TRP channels are activated by diacylglycerol (DAG) or its metabolites (polyunsaturated fatty acids), possibly in combination with the reduction in phosphatidyl inositol 4,5 bisphosphate (PIP2). Consistent with this view, diacylglycerol kinase is identified as a key enzyme required for response termination. Signaling is critically dependent upon efficient PIP2 synthesis; mutants of this pathway in combination with genetically targeted PIP2 reporters provide unique insights into the kinetics and regulation of PIP2 turnover. Recent evidence indicates that a growing number of mammalian TRP homologues are also regulated by lipid messengers, including DAG, arachidonic acid, and PIP2.
• The Contribution of Shaker K+ Channels to the Information Capacity of Drosophila Photoreceptors Nature. Feb, 2003  |   Pubmed ID: 12571596 An array of rapidly inactivating voltage-gated K+ channels is distributed throughout the nervous systems of vertebrates and invertebrates. Although these channels are thought to regulate the excitability of neurons by attenuating voltage signals, their specific functions are often poorly understood. We studied the role of the prototypical inactivating K+ conductance, Shaker, in Drosophila photoreceptors by recording intracellularly from wild-type and Shaker mutant photoreceptors. Here we show that loss of the Shaker K+ conductance produces a marked reduction in the signal-to-noise ratio of photoreceptors, generating a 50% decrease in the information capacity of these cells in fully light-adapted conditions. By combining experiments with modelling, we show that the inactivation of Shaker K+ channels amplifies voltage signals and enables photoreceptors to use their voltage range more effectively. Loss of the Shaker conductance attenuated the voltage signal and induced a compensatory decrease in impedance. Our results demonstrate the importance of the Shaker K+ conductance for neural coding precision and as a mechanism for selectively amplifying graded signals in neurons, and highlight the effect of compensatory mechanisms on neuronal information processing.
• Rescue of Light Responses in the Drosophila "null" Phospholipase C Mutant, NorpAP24, by the Diacylglycerol Kinase Mutant, RdgA, and by Metabolic Inhibition The Journal of Biological Chemistry. May, 2003  |   Pubmed ID: 12621055 Light responses in Drosophila are reportedly abolished in severe mutants of the phospholipase C (PLC) gene, norpA. However, on establishing the whole-cell recording configuration in photoreceptors of the supposedly null allele, norpAP24, we detected a small ( approximately 15 pA) inward current that represented spontaneous light channel activity. The current decayed during approximately 20 min, after which tiny residual responses (
• TRP Channels in Drosophila Photoreceptors: the Lipid Connection Cell Calcium. May-Jun, 2003  |   Pubmed ID: 12765684 The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP(2)). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein.
• Phototransduction: Shedding Light on Translocation Current Biology : CB. Sep, 2003  |   Pubmed ID: 14521858 Light induces the migration of arrestin to the photosensitive membrane in both vertebrate and invertebrate photoreceptors. New work has identified a phosphoinositide lipid binding domain in Drosophila arrestin and implicates PIP(3) in control of arrestin translocation.
• Regulation of Drosophila TRP Channels by Lipid Messengers Novartis Foundation Symposium. 2004  |   Pubmed ID: 15104181 In common with their vertebrate homologues, the prototypical Drosophila TRP channels are activated downstream of phospholipase C (PLC) by unknown mechanism(s). Most recent evidence in Drosophila photoreceptors now indicates that excitation is mediated, not by inositol 1,4,5-trisphosphate (IP3), but by lipid products of PLC action, such as diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids, PUFAs), or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). Compelling evidence for a PKC independent role of DAG comes from mutants of the rdgA gene, which encodes DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC or G protein. A role for PIP2 reduction is suggested by finding that conditions, which lead to acute PIP2 depletion--monitored by genetically targeted PIP2-sensitive ion channels--also lead to constitutive activation of TRP channels. Finally, recent data indicate that PUFAs activate TRP channels directly, and independently of PLC or metabolic inhibition. Together with evidence from several mammalian TRP homologues, these results suggest that regulation by lipids may be a defining feature of many TRP channels.
• 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) The Journal of Biological Chemistry. Nov, 2004  |   Pubmed ID: 15355960 The phosphatidylinositol 4,5-bisphosphate (PIP(2))-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP(2) levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP(2) concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP(2) and yielded channels generating currents proportional to the PIP(2) levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP(2) by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding approximately 150% of total microvillar phosphoinositides per second. PIP(2) was resynthesized with a half-time of approximately 50 s. When PIP(2) resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of approximately 40% loss of total PIP(2) per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP(2) levels were reduced approximately 5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant.
• Light Activation, Adaptation, and Cell Survival Functions of the Na+/Ca2+ Exchanger CalX Neuron. Feb, 2005  |   Pubmed ID: 15694324 In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. Here, we report that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel.
• Transient Receptor Potential-like Channels Are Essential for Calcium Signaling and Fluid Transport in a Drosophila Epithelium Genetics. Mar, 2005  |   Pubmed ID: 15695363 Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trpgamma in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRPgamma showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP(2b))-stimulated fluid transport rates were significantly reduced in tubules from the trpl(302) mutant and the trpl;trp double mutant, trpl(302);trp(343). However, a trp null, trp(343), had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca(2+)](i)) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl(302). Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP(2b)-stimulated fluid transport and calcium signaling. Rescue of trpl(302) with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl(302) was not compromised by introduction of the trp null, trp(343). Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules.
• Functional INAD Complexes Are Required to Mediate Degeneration in Photoreceptors of the Drosophila RdgA Mutant Journal of Cell Science. Apr, 2005  |   Pubmed ID: 15755798 The TRP family of ion channels mediates a wide range of calcium-influx phenomena in eukaryotic cells. Many members of this family are activated downstream of phosphoinositide hydrolysis but the subsequent steps that lead to TRP channel activation in vivo remain unclear. Recently, the lipid products of phosphoinositide hydrolysis (such as diacylglycerol and its metabolites) have been implicated in activating TRP channels in both Drosophila and mammals. In Drosophila photoreceptors, lack of diacylglycerol kinase (DGK) activity (encoded by rdgA) leads to both constitutive TRP-channel activity and retinal degeneration. In this study, using a novel forward-genetic screen, we identified InaD, a multivalent PDZ domain protein as a suppresser of retinal degeneration in rdgA mutants. We show that InaD suppresses rdgA and that the rescue is correlated with reduced levels of phospholipase Cbeta (PLCbeta), a key enzyme for TRP channel activation. Furthermore, we show that light, Gq and PLCbeta all modulate retinal degeneration in rdgA. The results demonstrate a previously unknown requirement for a balance of PLCbeta and DGK activity for retinal degeneration in rdgA. They also suggest a key role for the lipid products of phosphoinositide hydrolysis in the activation of TRP channels in vivo.
• Mechanisms of Light Adaptation in Drosophila Photoreceptors Current Biology : CB. Jul, 2005  |   Pubmed ID: 16005297 Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels. Ca(2+) influx via these channels is required for light adaptation, but although several molecular targets of Ca(2+)-dependent feedback have been identified, their contribution to adaptation is unclear. By manipulating cytosolic Ca(2+) via the Na(+)/Ca(2+) exchange equilibrium, we found that Ca(2+) inhibited the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca(2+) levels (0.1-10 microM Ca(2+)) and accurately mimicked light adaptation. However, PLC activity monitored with genetically targeted PIP(2)-sensitive ion channels (Kir2.1) was first inhibited by much higher (>/= approximately 50 microM) Ca(2+) levels, which occur only transiently in vivo. Ca(2+)-dependent inhibition of PLC, but not the LIC, was impaired in mutants (inaC) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca(2+)-dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca(2+) levels, whereas rapid inhibition of PLC by local Ca(2+) transients is required to terminate the response and ensures that PIP(2) reserves are not depleted during stimulation.
• Inhibition of Phospholipase C Activity in Drosophila Photoreceptors by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic Acid (BAPTA) and Di-bromo BAPTA Cell Calcium. Dec, 2005  |   Pubmed ID: 16140375 In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at "100 nM", approximately 10-fold inhibition at 100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 approximately 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 approximately 1mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (
• Calnexin is Essential for Rhodopsin Maturation, Ca2+ Regulation, and Photoreceptor Cell Survival Neuron. Jan, 2006  |   Pubmed ID: 16423697 In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration.
• Robustness of Neural Coding in Drosophila Photoreceptors in the Absence of Slow Delayed Rectifier K+ Channels The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2006  |   Pubmed ID: 16525044 Determining the contribution of a single type of ion channel to information processing within a neuron requires not only knowledge of the properties of the channel but also understanding of its function within a complex system. We studied the contribution of slow delayed rectifier K+ channels to neural coding in Drosophila photoreceptors by combining genetic and electrophysiological approaches with biophysical modeling. We show that the Shab gene encodes the slow delayed rectifier K+ channel and identify a novel voltage-gated K+ conductance. Analysis of the in vivo recorded voltage responses together with their computer-simulated counterparts demonstrates that Shab channels in Drosophila photoreceptors attenuate the light-induced depolarization and prevent response saturation in bright light. We also show that reduction of the Shab conductance in mutant photoreceptors is accompanied by a proportional drop in their input resistance. This reduction in input resistance partially restores the signaling range, sensitivity, and dynamic coding of light intensities of Shab photoreceptors to those of the wild-type counterparts. However, loss of the Shab channels may affect both the energy efficiency of coding and the processing of natural stimuli. Our results highlight the role of different types of voltage-gated K+ channels in the performance of the photoreceptors and provide insight into functional robustness against the perturbation of specific ion channel composition.
• Feedback Network Controls Photoreceptor Output at the Layer of First Visual Synapses in Drosophila The Journal of General Physiology. May, 2006  |   Pubmed ID: 16636201 At the layer of first visual synapses, information from photoreceptors is processed and transmitted towards the brain. In fly compound eye, output from photoreceptors (R1-R6) that share the same visual field is pooled and transmitted via histaminergic synapses to two classes of interneuron, large monopolar cells (LMCs) and amacrine cells (ACs). The interneurons also feed back to photoreceptor terminals via numerous ligand-gated synapses, yet the significance of these connections has remained a mystery. We investigated the role of feedback synapses by comparing intracellular responses of photoreceptors and LMCs in wild-type Drosophila and in synaptic mutants, to light and current pulses and to naturalistic light stimuli. The recordings were further subjected to rigorous statistical and information-theoretical analysis. We show that the feedback synapses form a negative feedback loop that controls the speed and amplitude of photoreceptor responses and hence the quality of the transmitted signals. These results highlight the benefits of feedback synapses for neural information processing, and suggest that similar coding strategies could be used in other nervous systems.
• TRP Channels and Lipids: from Drosophila to Mammalian Physiology The Journal of Physiology. Jan, 2007  |   Pubmed ID: 16990401 The transient receptor potential (TRP) ion channel family was the last major ion channel family to be discovered. The prototypical member (dTRP) was identified by a forward genetic approach in Drosophila, where it represents the transduction channel in the photoreceptors, activated downstream of a Gq-coupled PLC. In the meantime 29 vertebrate TRP isoforms are recognized, distributed amongst seven subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, TRPA, TRPN). They subserve a wide range of functions throughout the body, most notably, though by no means exclusively, in sensory transduction and in vascular smooth muscle. However, their precise physiological roles and mechanism of activation and regulation are still only gradually being revealed. Most TRP channels are subject to multiple modes of regulation, but a common theme amongst the TRPC/V/M subfamilies is their regulation by lipid messengers. Genetic evidence supports an excitatory role of diacylglycerol (DAG) for the dTRP's, although curiously only DAG metabolites (PUFAs) have been found to activate the Drosophila channels. TRPC2,3,6 and 7 are widely accepted as DAG-activated channels, although TRPC3 can also be regulated via a store-operated mechanism. More recently PIP2 has been shown to be required for activity of TRPV5, TRPM4,5,7 and 8, whilst it may inhibit TRPV1 and the dTRPs. Although compelling evidence for a direct interaction of DAG with the TRPC channels is lacking, mutagenesis studies have identified putative PIP2-interacting domains in the C-termini of several TRPV and TRPM channels.
• In Vivo Identification and Manipulation of the Ca2+ Selectivity Filter in the Drosophila Transient Receptor Potential Channel The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2007  |   Pubmed ID: 17234592 Null mutations in the transient receptor potential (trp) gene eliminate the major, Ca2+-selective component of the light-sensitive conductance in Drosophila photoreceptors. Although it is the prototypical member of the TRP ion channel superfamily, conclusive evidence that TRP is a pore-forming channel subunit in vivo is lacking. We show here that mutating a specific acidic residue (Asp621) in the putative pore virtually eliminated Ca2+ permeation in vivo and altered other biophysical properties of the native TRP conductance. The results identify Asp621 as a critical residue of the TRP Ca2+ selectivity filter, provide the first rigorous demonstration that a TRP protein is a pore-forming subunit in any native system, and point to the likely location of the pore in mammalian canonical TRP channels. The specific elimination of Ca2+ permeation in TRP also provided a unique opportunity to address the roles of Ca2+ influx in vivo. We found that Asp621 mutations profoundly affected several key aspects of the light response and caused light-dependent retinal degeneration.
• Vision: Dynamic Platforms Nature. Nov, 2007  |   Pubmed ID: 17972869
• Distinct Roles for Two Histamine Receptors (hclA and HclB) at the Drosophila Photoreceptor Synapse The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2008  |   Pubmed ID: 18632929 Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG "on" transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.
• Light-dependent Modulation of Shab Channels Via Phosphoinositide Depletion in Drosophila Photoreceptors Neuron. Aug, 2008  |   Pubmed ID: 18760696 The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.
• Ca2+-dependent Metarhodopsin Inactivation Mediated by Calmodulin and NINAC Myosin III Neuron. Sep, 2008  |   Pubmed ID: 18786361 Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination. M( *) was inactivated rapidly (tau approximately 20 ms) under control conditions, but approximately 10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M( *) inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M( *). Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.
• Regulation of Drosophila TRPC Channels by Lipid Messengers Cell Calcium. Jun, 2009  |   Pubmed ID: 19362736 The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, which are important components of calcium influx pathways in virtually all cells. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PIP(2) hydrolysis through a mechanism in which IP(3) receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that one or more PIP(2) generated lipid messengers, as well as PIP(2) itself, are essential for regulating TRP and TRPL activity. Evidence on the role of these lipid elements in regulating TRP and TRPL activity is discussed in this review.
• Lipids in Ca2+ Signalling--an Introduction Cell Calcium. Jun, 2009  |   Pubmed ID: 19406469 Lipids and lipid-derived metabolites are increasingly recognised as bonafide signalling molecules that regulate many cellular processes. These include the well-established InsP(3), diacylglycerol (DAG), PIP(2), PIP(3) and arachidonic acid (AA), as well as other poly-unsaturated fatty acids (PUFAs), lysophospholipids, sphingolipids, endocannabinoids and endovanilloids. They regulate a plethora of molecules that are involved in Ca(2+) signalling, including various ion channels, pumps and transporters, thereby triggering, modulating and fine-tuning Ca(2+) signals. Although appreciated individually, it seems timely to highlight the overall impact of lipids as signalling molecules and their role in Ca(2+) signalling, and this is the aim of this special issue of Cell Calcium.
• Phototransduction Motifs and Variations Cell. Oct, 2009  |   Pubmed ID: 19837030 Seeing begins in the photoreceptors, where light is absorbed and signaled to the nervous system. Throughout the animal kingdom, photoreceptors are diverse in design and purpose. Nonetheless, phototransduction-the mechanism by which absorbed photons are converted into an electrical response-is highly conserved and based almost exclusively on a single class of photoproteins, the opsins. In this Review, we survey the G protein-coupled signaling cascades downstream from opsins in photoreceptors across vertebrate and invertebrate species, noting their similarities as well as differences.
• Retinophilin is a Light-regulated Phosphoprotein Required to Suppress Photoreceptor Dark Noise in Drosophila The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2010  |   Pubmed ID: 20107052 Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.
• Activation of TRP Channels by Protons and Phosphoinositide Depletion in Drosophila Photoreceptors Current Biology : CB. Feb, 2010  |   Pubmed ID: 20116246 Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and TRPL) remains unresolved. In Drosophila, InsP(3) appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP(2). The fact that hydrolysis of the phosphodiester bond in PIP(2) by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context.
• Arrestin Translocation is Stoichiometric to Rhodopsin Isomerization and Accelerated by Phototransduction in Drosophila Photoreceptors Neuron. Sep, 2010  |   Pubmed ID: 20869596 Upon illumination, visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments, vertebrate outer segments or invertebrate rhabdomeres, where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant
• Metarhodopsin Control by Arrestin, Light-filtering Screening Pigments, and Visual Pigment Turnover in Invertebrate Microvillar Photoreceptors Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology. Mar, 2011  |   Pubmed ID: 21046112 The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment-arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.
• A Brief History of Trp: Commentary and Personal Perspective Pflugers Archiv : European Journal of Physiology. May, 2011  |   Pubmed ID: 21286746 The history of the discovery of the transient receptor potential (TRP) cation channel superfamily began in 1969 with Cosens and Manning's isolation of the Drosophila transient receptor potential mutant, in which the photoreceptor response decays during continuous illumination. Early studies from Minke found that the elementary light response was unaffected in trp mutants, and he attributed the defect to an intermediate stage of phototransduction. Montell and Rubin cloned the trp gene in 1989: they recognised it as a transmembrane protein, but also concluded that it did not encode the light-sensitive channels. In 1991, Minke and Selinger proposed that TRP represented a Ca2+ transporter required for refilling intracellular InsP3-sensitive Ca2+ stores, in turn required for activation of the light-sensitive channels. Also in 1991, after developing a photoreceptor patch clamp preparation, I showed that the light-sensitive channels themselves were highly permeable to Ca2+, questioning the need for such a dedicated Ca2+ transporter. In 1992, in collaboration with Minke, I resolved this paradox by showing there were two classes of light-sensitive channels, one highly Ca2+ permeable and eliminated in trp mutants. This represented the first and compelling evidence that TRP represented a light-sensitive channel and was supported by the cloning of the second light-sensitive channel, TRPL, by Kelly's lab. Three years later, in 1995, the labs of Montell and Birnbaumer independently cloned TRPC1, the first of 29 vertebrate TRP isoforms distributed amongst seven subfamilies.
• The INAD Scaffold is a Dynamic, Redox-regulated Modulator of Signaling in the Drosophila Eye Cell. Jun, 2011  |   Pubmed ID: 21703451 INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in the reduced state by raising the redox potential of its Cys606/Cys645 disulfide bond by ∼330 mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.
• The Drosophila SK Channel (dSK) Contributes to Photoreceptor Performance by Mediating Sensitivity Control at the First Visual Network The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2011  |   Pubmed ID: 21957252 The contribution of the SK (small-conductance calcium-activated potassium) channel to neuronal functions in complex circuits underlying sensory processing and behavior is largely unknown in the absence of suitable animal models. Here, we generated a Drosophila line that lacks the single highly conserved SK gene in its genome (dSK). In R1-R6 photoreceptors, dSK encodes a slow Ca²⁺-activated K(+) current similar to its mammalian counterparts. Compared with wild-type, dSK(-) photoreceptors and interneurons showed accelerated oscillatory responses and adaptation. These enhanced kinetics were accompanied with more depolarized dSK(-) photoreceptors axons, assigning a role for dSK in network gain control during light-to-dark transitions. However, compensatory network adaptation, through increasing activity between synaptic neighbors, overcame many detriments of missing dSK current enabling dSK(-) photoreceptors to maintain normal information transfer rates to naturalistic stimuli. While demonstrating important functional roles for dSK channel in the visual circuitry, these results also clarify how homeostatically balanced network functions can compensate missing or faulty ion channels.
• XPORT-dependent Transport of TRP and Rhodopsin Neuron. Nov, 2011  |   Pubmed ID: 22099462 TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem in the successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.
• Polarization Vision: Drosophila Enters the Arena Current Biology : CB. Jan, 2012  |   Pubmed ID: 22240470 Two new studies introduce the power of Drosophila genetics to polarization vision, revealing distinct photoreceptor inputs to polarotactic behaviour mediated by dorsal and ventral eye regions.
• Multiple Spectral Inputs Improve Motion Discrimination in the Drosophila Visual System Science (New York, N.Y.). May, 2012  |   Pubmed ID: 22605779 Color and motion information are thought to be channeled through separate neural pathways, but it remains unclear whether and how these pathways interact to improve motion perception. In insects, such as Drosophila, it has long been believed that motion information is fed exclusively by one spectral class of photoreceptor, so-called R1 to R6 cells; whereas R7 and R8 photoreceptors, which exist in multiple spectral classes, subserve color vision. Here, we report that R7 and R8 also contribute to the motion pathway. By using electrophysiological, optical, and behavioral assays, we found that R7/R8 information converge with and shape the motion pathway output, explaining flies' broadly tuned optomotor behavior by its composite responses. Our results demonstrate that inputs from photoreceptors of different spectral sensitivities improve motion discrimination, increasing robustness of perception.
• Stochastic, Adaptive Sampling of Information by Microvilli in Fly Photoreceptors Current Biology : CB. Aug, 2012  |   Pubmed ID: 22704990 In fly photoreceptors, light is focused onto a photosensitive waveguide, the rhabdomere, consisting of tens of thousands of microvilli. Each microvillus is capable of generating elementary responses, quantum bumps, in response to single photons using a stochastically operating phototransduction cascade. Whereas much is known about the cascade reactions, less is known about how the concerted action of the microvilli population encodes light changes into neural information and how the ultrastructure and biochemical machinery of photoreceptors of flies and other insects evolved in relation to the information sampling and processing they perform.
• Regulation of Arrestin Translocation by Ca2+ and Myosin III in Drosophila Photoreceptors The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2012  |   Pubmed ID: 22764229 Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, we show that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ∼100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabeling revealed that Arr2 in the cell body colocalized with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. We propose that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction.
• Photomechanical Responses in Drosophila Photoreceptors Science (New York, N.Y.). Oct, 2012  |   Pubmed ID: 23066080 Phototransduction in Drosophila microvillar photoreceptor cells is mediated by a G protein-activated phospholipase C (PLC). PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading by an unknown mechanism to activation of the prototypical transient receptor potential (TRP) and TRP-like (TRPL) channels. We found that light exposure evoked rapid PLC-mediated contractions of the photoreceptor cells and modulated the activity of mechanosensitive channels introduced into photoreceptor cells. Furthermore, photoreceptor light responses were facilitated by membrane stretch and were inhibited by amphipaths, which alter lipid bilayer properties. These results indicate that, by cleaving PIP(2), PLC generates rapid physical changes in the lipid bilayer that lead to contractions of the microvilli, and suggest that the resultant mechanical forces contribute to gating the light-sensitive channels.
• Common Mechanisms Regulating Dark Noise and Quantum Bump Amplification in Drosophila Photoreceptors Journal of Neurophysiology. Apr, 2013  |   Pubmed ID: 23365183 Absolute visual thresholds are limited by "dark noise," which in Drosophila photoreceptors is dominated by brief (∼10 ms), small (∼2 pA) inward current events, occurring at ∼2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the Gq α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca(2+) in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. Our results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca(2+)-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency.
• PI(4,5)P2 Depletion Underlies Retinal Degeneration in Drosophila Trp Mutants Journal of Cell Science. Feb, 2013  |   Pubmed ID: 23378018 The prototypical Transient Receptor Potential (TRP) channel is the major light-sensitive, and Ca(2+) permeable channel in Drosophila's microvillar photoreceptors. TRP channels are activated following hydrolysis of phosphatidyl-inositol 4,5 bisphosphate (PIP(2)) by the key effector enzyme phospholipase C (PLC). Mutants lacking TRP channels undergo light-dependent retinal degeneration, as a consequence of the reduced Ca(2+) influx. It has been proposed that degeneration is caused by defects in the Ca(2+) dependent visual pigment cycle, which result in accumulation of toxic phosphorylated metarhodopsin-arrestin complexes (M(PP)-Arr2). Here we show that two interventions, which prevent accumulation of M(PP)-Arr2, namely rearing under red light or eliminating the C-terminal rhodopsin phosphorylation sites, both failed to rescue degeneration in trp mutants. Instead degeneration in trp mutants reared under red light was rescued by mutation of PLC. Degeneration correlated closely with the light-induced depletion of PIP(2) that occurs in trp mutants due to failure of Ca(2+) dependent inhibition of PLC. Severe retinal degeneration was also induced in the dark in otherwise wild-type flies by overexpression of a bacterial PIP(2) 4'-phosphatase (SigD) to deplete PIP(2). In degenerating trp photoreceptors, phosphorylated Moesin, a PIP(2)-regulated membrane-cytoskeleton linker essential for normal microvillar morphology, delocalizes from the rhabdomere and there is extensive microvillar actin depolymerization. The results suggest the compromised light-induced Ca(2+) influx due to loss of TRP channels leads to PIP(2) depletion, resulting in dephosphorylation of Moesin, actin depolymerization and disintegration of photoreceptor structure.
• Fractional Ca(2+) Currents Through TRP and TRPL Channels in Drosophila Photoreceptors Biophysical Journal. May, 2013  |   Pubmed ID: 23663833 Light responses in Drosophila photoreceptors are mediated by two Ca(2+) permeable cation channels, transient receptor potential (TRP) and TRP-like (TRPL). Although Ca(2+) influx via these channels is critical for amplification, inactivation, and light adaptation, the fractional contribution of Ca(2+) to the currents (Pf) has not been measured. We describe a slow (τ ∼ 350 ms) tail current in voltage-clamped light responses and show that it is mediated by electrogenic Na(+)/Ca(2+) exchange. Assuming a 3Na:1Ca stoichiometry, we derive empirical estimates of Pf by comparing the charge integrals of the exchanger and light-induced currents. For TRPL channels, Pf was ∼17% as predicted by Goldman-Hodgkin-Katz (GHK) theory. Pf for TRP (29%) and wild-type flies (26%) was higher, but lower than the GHK prediction (45% and 42%). As predicted by GHK theory, Pf for both channels increased with extracellular [Ca(2+)], and was largely independent of voltage between -100 and -30 mV. A model incorporating intra- and extracellular geometry, ion permeation, diffusion, extrusion, and buffering suggested that the deviation from GHK predictions was largely accounted for by extracellular ionic depletion during the light-induced currents, and the time course of the Na(+)/Ca(2+) exchange current could be used to obtain estimates of cellular Ca(2+) buffering capacities.
• Photosensitive TRPs Handbook of Experimental Pharmacology. 2014  |   Pubmed ID: 24961970 The Drosophila "transient receptor potential" channel is the prototypical TRP channel, belonging to and defining the TRPC subfamily. Together with a second TRPC channel, trp-like (TRPL), TRP mediates the transducer current in the fly's photoreceptors. TRP and TRPL are also implicated in olfaction and Malpighian tubule function. In photoreceptors, TRP and TRPL are localised in the ~30,000 packed microvilli that form the photosensitive "rhabdomere"-a light-guiding rod, housing rhodopsin and the rest of the phototransduction machinery. TRP (but not TRPL) is assembled into multimolecular signalling complexes by a PDZ-domain scaffolding protein (INAD). TRPL (but not TRP) undergoes light-regulated translocation between cell body and rhabdomere. TRP and TRPL are also found in photoreceptor synapses where they may play a role in synaptic transmission. Like other TRPC channels, TRP and TRPL are activated by a G protein-coupled phospholipase C (PLCβ4) cascade. Although still debated, recent evidence indicates the channels can be activated by a combination of PIP2 depletion and protons released by the PLC reaction. PIP2 depletion may act mechanically as membrane area is reduced by cleavage of PIP2's bulky inositol headgroup. TRP, which dominates the light-sensitive current, is Ca(2+) selective (P Ca:P Cs >50:1), whilst TRPL has a modest Ca(2+) permeability (P Ca:P Cs ~5:1). Ca(2+) influx via the channels has profound positive and negative feedback roles, required for the rapid response kinetics, with Ca(2+) rapidly facilitating TRP (but not TRPL) and also inhibiting both channels. In trp mutants, stimulation by light results in rapid depletion of microvillar PIP2 due to lack of Ca(2+) influx required to inhibit PLC. This accounts for the "transient receptor potential" phenotype that gives the family its name and, over a period of days, leads to light-dependent retinal degeneration. Gain-of-function trp mutants with uncontrolled Ca(2+) influx also undergo retinal degeneration due to Ca(2+) cytotoxicity. In vertebrate retina, mice knockout studies suggest that TRPC6 and TRPC7 mediate a PLCβ4-activated transducer current in intrinsically photosensitive retinal ganglion cells, expressing melanopsin. TRPA1 has been implicated as a "photo-sensing" TRP channel in human melanocytes and light-sensitive neurons in the body wall of Drosophila.
• Phototransduction in Drosophila Current Opinion in Neurobiology. Oct, 2015  |   Pubmed ID: 25638280 Phototransduction in Drosophila's microvillar photoreceptors is mediated by phospholipase C (PLC) resulting in activation of two distinct Ca(2+)-permeable channels, TRP and TRPL. Here we review recent evidence on the unresolved mechanism of their activation, including the hypothesis that the channels are mechanically activated by physical effects of PIP2 depletion on the membrane, in combination with protons released by PLC. We also review molecularly explicit models indicating how Ca(2+)-dependent positive and negative feedback along with the ultracompartmentalization provided by the microvillar design can account for the ability of fly photoreceptors to respond to single photons 10-100× more rapidly than vertebrate rods, yet still signal under full sunlight.
• Speed and Sensitivity of Phototransduction in Drosophila Depend on Degree of Saturation of Membrane Phospholipids The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2015  |   Pubmed ID: 25673862 Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.
• In Vivo Tracking of Phosphoinositides in Drosophila Photoreceptors Journal of Cell Science. Dec, 2015  |   Pubmed ID: 26483384 In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, Tb(R332H) (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca(2+)-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca(2+)- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle.
• Professor Matti Weckström (1959-2015) Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology. Feb, 2016  |   Pubmed ID: 26749198
• Evidence for Dynamic Network Regulation of Drosophila Photoreceptor Function from Mutants Lacking the Neurotransmitter Histamine Frontiers in Neural Circuits. 2016  |   Pubmed ID: 27047343 Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdc (JK910) mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdc (JK910) photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdc (JK910) photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdc (JK910) R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdc (JK910) mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.
• Fly Photoreceptors Encode Phase Congruency PloS One. 2016  |   Pubmed ID: 27336733 More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli.
• Calcium Signalling in Drosophila Photoreceptors Measured with GCaMP6f Cell Calcium. Jul, 2017  |   Pubmed ID: 28238353 Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca(2+) influx in response to light, but whether Ca(2+) is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca(2+) signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with ∼1200 effectively absorbed photons and saturated (ΔF/F0∼10-20) with 10000-30000 photons. In Ca(2+) free bath, smaller (ΔF/F0 ∼4), long latency (∼200ms) light-induced Ca(2+) rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na(+) was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca(2+) free rises were also eliminated in Na(+)/Ca(2+) exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca(2+) free rises are strictly dependent on Na(+) influx and activity of the exchanger, suggesting they reflect re-equilibration of Na(+)/Ca(2+) exchange across plasma or intracellular membranes following massive Na(+) influx. Any tiny Ca(2+) free rise remaining without exchanger activity was equivalent to