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B Minke

Publications and source records attributed to B Minke.

At least 37 records · Page 2Linked to original sources
PubMed1994

Calcium-dependent inactivation of light-sensitive channels in Drosophila photoreceptors.

Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light-sensitive channels activate spontaneously, generating a "rundown current" (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents which inactivate increasingly rapidly as Ca2+ is raised. On depolarization inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild-type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition (12 ms with 1.5 mM external Ca2+ at -60 mV) is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induced transient currents which appeared similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivated more rapidly with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached.

Animals↗
PubMed1994

Genetic dissection of light-induced Ca2+ influx into Drosophila photoreceptors.

Invertebrate photoreceptors use the inositol-lipid signaling cascade for phototransduction. A useful approach to dissect this pathway and its regulation has been provided by the isolation of Drosophila visual mutants. We measured extracellular changes of Ca2+ [delta Ca2+]o in Drosophila retina using Ca(2+)-selective microelectrodes in both the transient receptor potential (trp) mutant, in which the calcium permeability of the light-sensitive channels is greatly diminished and in the inactivation-but-no-afterpotential C (inaC) mutant which lacks photoreceptor-specific protein kinase C (PKC). Illumination induced a decrease in extracellular [Ca2+] with kinetics and magnitude that changed with light intensity. Compared to wild-type, the light-induced decrease in [Ca2+]o (the Ca2+ signal) was diminished in trp but significantly enhanced in inaC. The enhanced Ca2+ signal was diminished in the double mutant inaC;trp indicating that the effect of the trp mutation overrides the enhancement observed in the absence of eye-PKC. We suggest that the decrease in [Ca2+]o reflects light-induced Ca2+ influx into the photoreceptors and that the trp mutation blocks a large fraction of this Ca2+ influx, while the absence of eye specific PKC leads to enhancement of light-induced Ca2+ influx. This suggestion was supported by Ca2+ measurements in isolated ommatidia loaded with the fluorescent Ca2+ indicator, Ca Green-5N, which indicated an approximately threefold larger light-induced increase in cellular Ca2+ in inaC relative to WT. Our observations are consistent with the hypothesis that TRP is a light activated Ca2+ channel and that the increased Ca2+ influx observed in the absence of PKC is mediated mainly via the TRP channel.

Animals↗
PubMed1993

Ca2+ limits the development of the light response in Drosophila photoreceptors.

The development of the light response was followed in Drosophila photoreceptors at 25 degrees C. In whole-cell recordings from dissociated ommatidia, responses to light were first detected at 82 h post-puparium formation; over the next 8 h sensitivity to light increased exponentially by 5 or 6 orders of magnitude. The end of this phase coincided with the maturation of the rhabdomere as measured by whole-cell capacitance. There was a modest 5-10fold further increase in sensitivity over the final 10 h of pupal development (90-100 h). During a narrow developmental time window (82-87 h) no responses could be detected using non-invasive recording techniques (electroretinogram or suction electrode), and responses to light could only be elicited in whole-cell recordings when micromolar concentrations of Ca2+ are included in the pipette. It seems unlikely that cytosolic Ca2+ per se is the limiting factor, and we suggest instead that the failure to respond to light is due to the lack of Ca2+ in the InsP3-sensitive intracellular stores and that the presence of Ca2+ in these stores is an absolute requirement for phototransduction in Drosophila.

Animals↗
PubMed1993

Protein kinase C is required for light adaptation in Drosophila photoreceptors.

Protein kinase C (PKC) is a key enzyme for many cellular processes but its physiological roles are poorly understood. An excellent opportunity to investigate the function of PKC has been provided by the identification of an eye-specific PKC in Drosophila and a null PKC mutant, inaCP209 (refs 5,6). Bright conditioning lights delivered to inaC photoreceptors lead to an abnormal loss of sensitivity in whole cell recordings from dissociated ommatidia; this has been interpreted as 'hyper-adaptation' and PKC's role has been suggested to be distinct from light adaptation. A presumably related finding is that during intense light, the response of inaC declines to baseline. Invertebrate photoreceptors use the phosphoinositide signalling cascade, responding to single photons with so-called quantum bumps which sum to form the macroscopic response to light. Light adaptation allows photoreceptors to adjust their sensitivity over the enormous range of ambient intensities. Although the molecular mechanism of light adaptation remains obscure, it is a negative-feedback process mediated by a rise in cytosolic calcium and a decrease in bump size. We now show that under physiological conditions light adaptation is severely reduced in inaC, suggesting that eye-specific PKC, itself activated by a rise in cytosolic calcium and diacylglycerol, is required for adaptation. Furthermore, we show that in the absence of PKC individual bumps fail to terminate normally, an effect that can account for the pleiotropic manifestations of the inaC phenotype.

Adaptation, Ocular↗
PubMed1993

Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.

Animals↗
PubMed1993

Novel Ca2+ channels underlying transduction in Drosophila photoreceptors: implications for phosphoinositide-mediated Ca2+ mobilization.

Drosophila photoreceptors are excellent models for studies of the ubiquitous phosphoinositide signalling cascade. Recent studies suggest that light-induced phosphoinositide hydrolysis in Drosophila leads to the activation of two classes of channels. One is selective for Ca2+ and absent in the transient receptor potential mutant trp. The trp gene product, which shows some structural similarity to vertebrate voltage-gated Ca2+ channels, may thus define a novel family of second-messenger-operated Ca2+ channels generally responsible for the widespread but poorly understood phenomenon of phosphoinositide-mediated Ca2+ entry. The other channel is a non-selective cation channel that requires Ca2+ for activation. As well as being a major charge carrier for the light-induced current, Ca2+ influx via the trp-dependent channels appears to be required for refilling Ca2+ stores sensitive to inositol 1,4,5-trisphosphate and for feedback regulation (light adaptation) of the transduction cascade.

Animals↗
PubMed1992

Characterization of fly rhodopsin kinase.

Rhodopsin kinase activity of Musca domestica was characterized in a reconstitution assay, using urea-treated eye membranes as substrate and a purified fraction of eye cytosol as the enzyme. Analysis of kinase activity in fly eye, brain and abdomen extracts by reconstitution assays revealed that fly rhodopsin kinase is an eye-specific enzyme. It preferentially phosphorylates the light-activated form of rhodopsin (metarhodopsin) and has little activity with other protein substrates. Rhodopsin kinase binds to metarhodopsin and is released from rhodopsin-containing membranes. Metarhodopsin is a poor substrate for kinases from tissues other than the eye, making it a unique substrate for rhodopsin kinase. Rhodopsin kinase is inhibited by heparin, but not by the protein inhibitor of cAMP-dependent protein kinase. Its Km for ATP is 9 microM. Since fly rhodopsin is coupled to phospholipase C, studies of the interaction of rhodopsin with rhodopsin kinase can be useful in analysis of the reactions that lead to termination of the inositol-phospholipid-signaling pathway.

Animals↗
PubMed1992

Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.

Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant.

Action Potentials↗
PubMed1992

Calcium is necessary for light excitation in barnacle photoreceptors.

Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca2+ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La3+), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca2+ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca2+, but not K+ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La3+. The EGTA experiments suggest that internal Ca2+ is necessary for light excitation of barnacle photoreceptors while the La3+ experiments suggest that La(3+)-sensitive inward current is necessary to maintain excitation during prolonged light.

Animals↗
PubMed1992

The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors.

Invertebrate phototransduction is an important model system for studying the ubiquitous inositol-lipid signaling system. In the transient receptor potential (trp) mutant, one of the most intensively studied transduction mutants of Drosophila, the light response quickly declines to baseline during prolonged intense light. Using whole-cell recordings from Drosophila photoreceptors, we show that the wild-type response is mediated by at least two functionally distinct classes of light-sensitive channels and that both the trp mutation and a Ca2+ channel blocker (La3+) selectively abolish one class of channel with high Ca2+ permeability. Evidence is also presented that Ca2+ is necessary for excitation and that Ca2+ depletion mimics the trp phenotype. We conclude that the recently sequenced trp protein represents a class of light-sensitive channel required for inositide-mediated Ca2+ entry and suggest that this process is necessary for maintained excitation during intense illumination in fly photoreceptors.

Animals↗
PubMed1992

The nss mutation or lanthanum inhibits light-induced Ca2+ influx into fly photoreceptors.

Ion-selective calcium microelectrodes were inserted into the compound eyes of the wild-type sheep blowfly Lucilia or into the retina of the no steady state (nss) mutant of Lucilia. These electrodes monitored light-induced changes in the extracellular concentration of calcium (delta[Ca2+]o) together with the extracellularly recorded receptor potential. Prolonged dim lights induced a steady reduction in [Ca2+]o during light in the retina of normal Lucilia, while relatively little change in [Ca2+]o was observed in the retina of the nss mutant. Prolonged intense light induced a multiphasic change in [Ca2+]o: the [Ca2+]o signal became transient, reaching a minimum within 6 s after light onset, and then rose to a nearly steady-state phase below the dark concentration. When lights were turned off, a rapid increase in [Ca2+]o was observed, reaching a peak above the dark level and then declining again to the dark level within 1 min. In analogy to similar studies conduced in the honeybee drone, we suggest that the reduction in [Ca2+]o reflects light-induced Ca2+ influx into the photoreceptors, while the subsequent increase in [Ca2+]o reflects the activation of the Na-Ca exchange which extrudes Ca2+ from the cells. In the nss mutant in response to intense prolonged light, the receptor potential declines to baseline during light while the Ca2+ signal is almost abolished, revealing only a short transient reduction in [Ca2+]o. Application of lanthanum (La3+), but not nickel (Ni2+), into the retinal extracellular space of normal Lucilia mimicked the effect of the nss mutation on the receptor potential, while complete elimination of the Ca2+ signal in a reversible manner was observed. The results suggest that La3+ and the nss mutation inhibit light-induced Ca2+ influex into the photoreceptor in a manner similar to the action of the trp mutation in Drosophila, which has been shown to block specifically a light-activated Ca2+ channel necessary to maintain light excitation.

Animals↗
PubMed1991

Lanthanum reduces the excitation efficiency in fly photoreceptors.

Lanthanum (La3+), a known inhibitor of Ca2+ binding proteins, was applied to the extracellular space of fly retina. Shot noise analysis indicated that a combination of intense light and La3+ caused a large (down to zero) reduction in the rate of occurrence of the quantal responses to single photons (quantum bumps) which sum to produce the photoreceptor potential. Light in the presence of La3+ also increased the effective bump duration. These effects are very similar to the effects of the mutations trp of Drosophila and nss of Lucilia flies on the quantum bump rate and duration. La3+ applied to the nss mutant caused only a small reduction in the bump rate, suggesting that La3+ may affect the nss gene product which is deficient in the mutant. The close similarity in the properties of the receptor potential of the La(3+)-treated photoreceptor of the wild type and of the nss mutant together with existing evidence for the highly reduced intracellular Ca2+ ([Ca2+]i) level in nss photoreceptors suggest that both La3+ and the mutation cause a severe reduction in [Ca2+]i. This effect may arise from an inhibition of a Ca2+ transporter protein located in the surface membrane that normally replenishes Ca2+ pools in the photoreceptors, a process essential for light excitation.

Animals↗
PubMed1991

Spatial restriction of light adaptation and mutation-induced inactivation in fly photoreceptors.

The spatial spread within fly photoreceptors of 2 forms of desensitization by bright light have been investigated: the natural process of light adaptation in normal Musca photoreceptors and a receptor-potential inactivation in the no-steady-state (nss) mutant of the sheep blowfly Lucilia. The suction-electrode method used for recording from vertebrate rods was applied to fly ommatidia. A single ommatidium in vitro was partially sucked into a recording pipette. Illumination of the portion of the ommatidium within the pipette resulted in a flow of current having a wave form similar to that of the receptor potential and polarity consistent with current flow into the illuminated region of the photoreceptors. Two 5-microns slits of light, positioned at right angles to the ommatidial axis, were employed to determine the spread of light adaptation or inactivation along the ommatidium. The intensity of a flash of light delivered to one (adapting) slit was adjusted until it produced a criterion fractional reduction in the response to the other (test) slit. The reciprocal of this intensity of the adapting slit was taken as a measure of the effectiveness of the slit in causing light adaptation or inactivation. The effectiveness of the slit in causing light adaptation in normal Musca ommatidia fell as the adapting and test slits were moved farther apart along the ommatidial axis, declining to half its maximal value at a distance of 13 +/- 2 microns. Similar measurements of the effectiveness of a slit in causing light-induced inactivation in the nss mutant of Lucilia also demonstrated localization, declining to half its maximal value at a distance between the slits of 9 +/- 1 microns. Neither light adaptation nor inactivation by the nss mutation, therefore, appear to be mediated by voltage or by a highly diffusible agent. The results are consistent with the idea that inactivation by the nss mutation replaces adaptation in the mutant photoreceptors.

Adaptation, Physiological↗
PubMed1990

Phorbol ester induces photoreceptor-specific degeneration in a Drosophila mutant.

In the retinal degeneration B (rdgB) mutant of Drosophila, the major class of photoreceptors degenerate when the fly is raised in the light for several days; raising the fly in the dark largely prevents the degeneration. Thus, the rdgB is a conditional mutant that requires the operation of some stages of the phototransduction cascade to express its characteristic phenotype. We report here experiments that examine the ability of chemical agents to mimic light by causing photoreceptor-specific degeneration in the dark. Application of a specific activator of protein kinase C, phorbol ester, to eyes of rdgB flies led to a degeneration of the photoreceptors that was indistinguishable from that caused by light: both light and phorbol ester-induced degeneration were characterized by (i) selective degeneration of one class of photoreceptors; (ii) a unique pattern of degeneration; and (iii) the appearance of light-induced regenerative spikes at early stages of degeneration. Application of phorbol ester to the eyes of wild-type flies had no effect. We suggest that light or phorbol ester activates a protein kinase C and results in a sustained or excessive phosphorylation of proteins in the rdgB mutant, leading to photoreceptor degeneration. Furthermore, the results are consistent with identification of the rdgB gene product as a phosphoprotein phosphatase that is nonfunctional or absent in the mutant.

Animals↗
PubMed1989

Light-induced retinal degeneration in rdgB (retinal degeneration B) mutant of Drosophila: electrophysiological and morphological manifestations of degeneration.

Quantitative light and electron microscopy was used to monitor the extent of retinal degeneration as a function of age and temperature in the white-eyed rdgBKS222 mutant of Drosophila melanogaster. Parallel measurements of the electroretinogram (ERG) of the degenerating retina reveal a new phenomenon--the appearance of spike potentials following illumination with bright light. These spikes, which do not appear in the normal fly retina, have a relatively long duration (20-50 ms), regenerative properties, and a rate of occurrence which increases with increasing light intensity. The spikes differed from the light response in being more susceptible to CO2 and to cuts in the eye. The spikes completely disappeared at low extracellular Ca2+ levels which did not reduce the amplitude of the light response. The spike potentials become triphasic when the recording electrode is advanced to the level of the basement membrane. This suggests that the spike potentials originate from the photoreceptor axons as a result of synchronous opening of voltage-dependent channels in a large number of photoreceptor cells. The occurrence of spike potentials during the process of degeneration was studied. Two pahses can be distinguished: (1) Spike potentials appear in retinae of 2-3-day-old flies which display few morphological signs of degeneration. The frequency of appearance of spike potentials decreases in retinae of 14-16-day-old flies which show extensive degeneration of the R1-6 photoreceptor cells but no degeneration of the central R7,8 cells. (2) Spike potentials appear more frequently again in flies of 22-24 d of age. This is probably a consequence of degeneration of the remaining R7,8 photoreceptor cells. Temperature and the light-dark cycle had a critical effect on degeneration. Eight-day-old mutants raised at 19 degrees C in a normal light-dark cycle showed only little degeneration. Eight-day-old mutants raised at 24 degrees C showed only a slight degeneration when raised in the dark. However, the degree of degeneration was greatly enhanced in the mutants raised at 24 degrees C under a light-dark cycle regime. The combined electrophysiological and morphological study of the degeneration, as a function of age and temperature, revealed that (1) the degeneration process takes place even in darkness, but at a slow rate, while light greatly accelerates the degeneration. (2) The degeneration is negligible at 19 degrees C, even during light, in the first week after eclosion.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1989

Chemically induced retinal degeneration in the rdgB (retinal degeneration B) mutant of Drosophila.

Chemicals which affect different steps of the phototransduction cascade were used to identify the site of action of the rdgB gene product of Drosophila. In the rdgB mutant, the photoreceptor cells degenerate after several days of exposure to light, whereas raising the flies in the dark largely prevents the degeneration. In the rdgBKS222 mutant, which was used in the present studies, the light-induced degeneration is characterized by (1) selective degeneration of the peripheral but not the central photoreceptor cells; (2) random distribution of degenerated cells among ommatidia; and (3) the degeneration is specific to the rdgB but not the wild-type photoreceptor cells. In the present study, we show that application of specific chemical agents to the eyes of rdgBKS222 flies in the dark mimics the effects of light and causes retinal degeneration indistinguishable from light. The agents used in these studies are the metabolically stable GTP analogs GTP gamma S and Gpp(NH)p as well as fluoride ions, which are known to activate the transducing guanine nucleotide binding protein (G-protein of fly photoreceptors). It is unlikely that the chemically induced retinal degeneration is mediated by effects on energy metabolism, since application of the metabolic inhibitors CN- and 2-deoxy-D-glucose did not increase the extent of retinal degeneration over that observed in control flies treated with Ringer solution. The GDP analog GDP beta S, which inhibits G-protein activity, greatly reduced the extent of retinal degeneration in the dark, over that observed in control flies treated with Ringer solution. These results suggest that activation of the G-protein precedes the step in the transduction cascade that leads to retinal degeneration and provides a powerful tool to investigate the molecular mechanism of light-induced degeneration in the rdgB mutant.

Animals↗