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R C Hardie

Publications and source records attributed to R C Hardie.

At least 19 recordsLinked to original sources
PubMed1997

Functional equivalence of native light-sensitive channels in the Drosophila trp301 mutant and TRPL cation channels expressed in a stably transfected Drosophila cell line.

Drosophila photoreceptors express two putative cation channels encoded by the transient receptor potential (trp) and trp-like (trpl) genes, which represent prototypical members of a novel family of phosphoinositide-regulated calcium influx channels. Mutations of both trp and trpl selectively abolish components of the light-sensitive current and, when heterologously expressed, both generate cation permeable conductances; however, a detailed comparison of recombinant and native channel properties is lacking. To more rigorously test the hypothesis that TRPL channels mediate one component of the light-sensitive current we have generated cell lines (Drosophila S2 cells) stably transfected with trpl cDNA and compared the recombinant channel properties with those of the light-sensitive conductance in situ in a Drosophila trp mutant under identical conditions. We found close correspondence in respect of a number of quantifiable biophysical parameters including: current voltage relationships, ionic selectivity, voltage independent block by external Mg2+ ions and effective single channel conductance and gating kinetics derived by noise analysis. Our estimate of 60-70 pS for channel conductance was confirmed directly in patch clamp recordings of single TRPL channels in S2 cells. These findings indicate that channels encoded by the trpl gene can completely account for the component of the light-sensitive conductance remaining in the trp mutant.

Animals↗
PubMed1997

In vivo analysis of the drosophila light-sensitive channels, TRP and TRPL.

We have tested the proposal that the light-sensitive conductance in Drosophila is composed of two independent components by comparing the wild-type conductance with that in mutants lacking one or the other of the putative light-sensitive channel subunits, TRP and TRPL. For a wide range of cations, ionic permeability ratios in wild type were always intermediate between those of trp and trpl mutants. Effective channel conductances derived by noise analysis in wild type were again intermediate (17 pS; c.f. 35 pS in trp and 4 pS in trpl) and also showed a complex voltage dependence, which was quantitatively explained by the summation of TRPL and TRP channels after taking their different reversal potentials into account. Although La3+ partially blocked the light response in wild-type photoreceptors, it increased the effective single channel conductance. The results indicate that the wild-type light-activated conductance is composed of two separate channels, with the properties of TRP- and TRPL-dependent channels as determined in the respective mutants.

Animals↗
PubMed1996

Calcium signalling: setting store by calcium channels.

Recent work, stemming from genetic studies of phototransduction in Drosophila, has identified a set of homologous channel proteins that appear to mediate capacitative Ca2+ entry in species from worms and files to humans.

Animals↗
PubMed1996

INDO-1 measurements of absolute resting and light-induced Ca2+ concentration in Drosophila photoreceptors.

Absolute Ca2+ levels in dissociated Drosophila photoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca2+ influx that saturated the dye (>10 microM Ca2+), but lagged the electrical response by 2.8 msec. Resting Ca2+ levels in the dark, measured during the latent period before the response, averaged 160 nM in normal Ringer's (1.5 mM Ca2+). Ca2+ increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca2+ during the latent period; and (2) by using ninaE mutants with greatly reduced rhodopsin levels. Ca2+ rose linearly as a function of the time integral of the light-sensitive current with a slope of 2.7 nM/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nM/pC, indicating a 2.5-fold reduction in the fractional Ca2+ current. From these data, it can also be estimated that >99% of the Ca2+ influx is effectively buffered by the cell. In Ca2+-free Ringer's, resting cytosolic Ca2+ was reduced (to 30-70 nM), but contrary to previous reports, significant light-induced increases (approximately 250 nM) could be elicited. This rise was reduced to <20 nM when extracellular Na+ was replaced with N-methyl-D-glucamine, suggesting that it could be attributed to Na+ influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nM.

Animals↗
PubMed1996

A quantitative estimate of the maximum amount of light-induced Ca2+ release in Drosophila photoreceptors.

Simultaneous measurements of the light-induced current (LIC) and cytosolic Ca2+ (using INDO-1) were made in Drosophila photoreceptors. In the presence of 1.5 mM Cao2+, the UV light used to measure INDO-1 fluorescence saturated the LIC and induced a large Ca2+ rise. In the absence of extracellular Ca2+ and with Na+ replaced by N-methyl-D-glucamine, the light-induced Ca2+ rise was virtually abolished. A residual rise of about 20 nM is regarded as an upper estimate of Ca2+ released from internal stores. To estimate the Ca2+ flux required to generate such a rise, Ca2+ influx signals in response to weak light steps (500 ms LED stimulus) were measured in the presence of external Ca2+. The relationship between [Ca(in)] and the total charge carried during the LIC had a slope of 2.7 nM pC-1. Assuming that 50% of the LIC is carried by Ca2+ and that the single-channel Ca2+ current carried by the InsP3 receptor is 0.04 pA, it was estimated that about 350 InsP3 receptors should have been sufficient to generate a Ca2+ rise of 20 nM within 500 ms. By contrast, the current activated by the UV measuring light was equivalent to the activation of at least 5000 quantum bumps, making it unlikely that InsP3-induced Ca2+ release could have been the causal event for excitation under these conditions.

Animals↗
PubMed1996

Excitation of Drosophila photoreceptors by BAPTA and ionomycin: evidence for capacitative Ca2+ entry?

It has been suggested that excitation in Drosophila photoreceptors may be mediated by the depletion of intracellular Ca2+ stores (capacitative Ca2+ entry). To investigate this hypothesis, simultaneous whole-cell recordings and Indo-1 Ca2+ measurements were made from dissociated Drosophila photoreceptors, whilst testing the effects of Ca2+ releasing agents. In Ca2+ free Ringer's solution, thapsigargin raised cytosolic Ca2+ by approximately 80 nM; subsequent application of ionomycin released further Ca2+ (approximately 100 nM). A possible third compartment was indicated by the ability of monensin to mobilize further Ca2+ after saturating doses of ionomycin. Under most conditions, none of these agents activated an inward conductance, and their effects on the light response were consistent with their effects on cytosolic Ca2+. However, in the absence of both external Ca2+ and Mg2+ (to relieve a Mg2+ block of the light-sensitive channels), and after loading cells with BAPTA buffering cytosolic free Ca2+ at approximately 10 nM, ionomycin (but not thapsigargin) activated inward currents of approximately 800 pA. The response to ionomycin was enhanced (10 nA) by buffering cytosolic Ca2+ at 250 nM. A similar current also developed after approximately 3 min in cells loaded with Ca-BAPTA without any ionomycin application. The current-voltage relationships of currents activated by Ca-BAPTA or ionomycin were indistinguishable from that of the light-activated conductance and were similarly affected by a null mutation of the transient receptor potential (trp) gene which is believed to encode a subunit of the light-sensitive channels. These experiments provide some evidence for the suggestion that the light-activated and trp-dependent conductance in Drosophila photoreceptors can be activated by depletion of internal stores. However, activation by Ca-BAPTA and ionomycin had an absolute requirement for cytosolic Ca2+ as no currents could be activated by ionomycin in cells loaded with BAPTA and no Ca2+.

Animals↗
PubMed1995

Effects of intracellular Ca2+ chelation on the light response in Drosophila photoreceptors.

In order to test the hypothesis that excitation in Drosophila photoreceptors is mediated by Ca2+ released from internal stores, the Ca2+ buffers EGTA, BAPTA and di-bromo-BAPTA (DBB) were introduced into dissociated photoreceptors via whole-cell recording pipettes. All buffers were preloaded with Ca2+ to provide the same free Ca2+ concentration (250 nM). EGTA (up to 18 mM free buffer) had only weak effects upon voltage-clamped flash responses in normal Ringer's solution (1.5 mM Ca (2 0+)), and no effect in Ca2+ -free solution. The maximum BAPTA concentration tested (14.4 mM free BAPTA) reduced the initial rate of rise by ca. 5000-fold in normal Ringer's solution; by ca. 500-fold in Ca2+ free solution; and only ca. 60-fold in the absence of Mg2+, which preferentially blocks one component of the light-sensitive current. Although BAPTA delayed the time-to-peak in normal Ringer's solution, responses in Ca2+ free Ringer's solution were accelerated. These results support the role of Ca2+ influx in regulating sensitivity and response kinetics; however, in view of the high concentrations required to attenuate responses in Ca2+ free Ringer's solution, the role of Ca2+ release in excitation remains unclear. DBB was ca. 2-3 fold more potent than BAPTA, and at concentrations > 5 mM had a qualitatively different action, greatly delaying the time-to-peak. This suggests DBB may have distinct pharmacological actions or access to compartments inaccessible to BAPTA. The only current activated by introducing 5-500 microM Ca2+ (buffered with nitrilo-triacetic acid) was electrogenic Na+/Ca2+ exchange. When this was blocked by removing Nao0+, a novel cationic conductance was activated. However, its properties did not resemble those the light-activated conductance, and thus do not support the hypothesis that Ca2+ is sufficient for excitation.

Animals↗
PubMed1995

Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp.

Drosphoinate photoreceptors, represent a paradigm for the genetic dissection of phototransduction and, more generally for Ca2+ signaling. As in most invertebrates, phototransduction in Drosophila is mediated by the phosphoinositide (PI) cascade and is completely blocked by null mutations of the norpA gene which encodes a phospholipase C-beta isoform. The light-activated conductance in Drosophila is normally highly permeable to Ca2+, but in null mutants of the trp gene Ca2+ permeability is greatly reduced. Furthermore, the trp gene sequence shows homologies with voltage gated Ca2+ channels, suggesting that trp encodes a light-sensitive channel subunit. Ca2+ influx via these channels is instrumental in light adaptation, and profoundly influences phototransduction via positive and negative feedback at multiple molecular targets including protein kinase C. The mechanism of activation of the light-sensitive channels remains unresolved. A requirement for Ca2+ release from internal stores is suggested by the finding that Drosophila photoreceptors cannot sustain a maintained response under various conditions which might be expected to result in depletion of Ca2+ stores. However, Ca2+ release cannot be detected by Ca2+ indicator dyes and raising Ca2+ by photorelease of caged Ca2+ fails to mimic excitation. Recent studies, both in situ and with heterologously expressed trp protein, suggest that the trp-dependent channels may be activated by a process analogous to 'capacitative Ca2+ entry', a widespread, but poorly understood mode of PI-regulated Ca2+ influx in vertebrate cells.

Animals↗
PubMed1995

Serotonin modulates the voltage dependence of delayed rectifier and Shaker potassium channels in Drosophila photoreceptors.

We describe the in situ modulation of potassium channels in a semi-intact preparation of the Drosophila retina. In whole-cell recordings of photoreceptors, rapidly inactivating Shaker channels are characterized by a conspicuously negative voltage operating range; together with a delayed rectifier, these channels are specifically modulated by the putative efferent neurotransmitter serotonin. Contrary to most potassium channel modulations, serotonin induced a reversible positive shift in the voltage operating range, of +30 mV for the Shaker channels and +10-14 mV for the delayed rectifier. The maximal current amplitudes were unaffected. Modulation was not affected by the subunit-specific Shaker mutations ShE62 and T(1;Y)W32 or a null mutation of the putative modulatory subunit eag. The modulation of both channels was mimicked by intracellularly applied GTP gamma S.

Animals↗
PubMed1995

Magnesium-dependent block of the light-activated and trp-dependent conductance in Drosophila photoreceptors.

1. The effect of Mg2+ on the light-sensitive conductance in Drosophila photoreceptors was examined with the use of whole cell voltage-clamp recordings from dissociated ommatidia. In wild type (WT) photoreceptors, at resting potential (-70 mV). Mgo2+ reduces response amplitude by up to approximately 4-fold in the presence of normal (1.5 mM) Cao2+ and by up to 20-fold in the absence of Cao2+. The Mg2+ concentration required for 50% maximum block (K1/2) was approximately 1 mM with 1.5 mM Cao2+ and approximately 280 microM in Ca(2+)-free Ringer. 2. The Mg2+ block was largely relieved in photoreceptors of the transient receptor potential mutant (trp): the maximum block being only approximately twofold with a K1/2 of approximately 4 mM in both Ca(2+)-free and 1.5 mM Cao2+. 3. The Mg2+ block in WT, but not in trp, was strongly voltage dependent, being relieved by both hyperpolarization and depolarization. The Mg2+ block in WT also resulted in slower response kinetics because of the associated decrease in Ca2+ influx. 4. Noise analysis indicates that, with normal Ca(zero)2+, the Mg2+ block in WT is associated with a approximately 10-fold reduction in effective single-channel conductance al resting potential. 5. The results support the hypothesis that the trp gene encodes a subunit of a light-sensitive channel, which is required for sensitivity to block by Mg2+. The concentration and voltage dependence of the Mg2+ block suggest it plays an important physiological role in determining the gain, kinetics, and signal-to-noise of transduction.

Animals↗
PubMed1995

Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors.

Substantial evidence implicates the phosphoinositide cascade in invertebrate phototransduction, but the final pathway of excitation remains obscure. In order to test the hypothesis that Ca2+ is the excitatory messenger rapid concentration jumps of cytosolic Ca2+ were achieved in dissociated Drosophila photoreceptors by flash photolysis of the caged Ca2+ compounds DM-nitrophen and nitr-5. Both compounds were introduced via patch pipettes used to record whole-cell currents. Calibrations using INDO-1 and Mag-INDO-1 indicated that photolysis of DM-nitrophen (5 mM loaded with 4 mM Ca2+), raised Ca, to ca. 20-50 microM, and nitr-5 (same loading) to ca. 1-2 microM. In mutants lacking light responses (ora, lacking rhodopsin; norpA, lacking phospholipase C; trp, which is inactivated by conditioning lights), the only current evoked by photolysis of DM-nitrophen was a small inward current with no detectable latency. This current did not reverse at +80 mV and was blocked by substitution of external Na+ for Li+, suggesting it represents activation of an electrogenic Na+/Ca2+ exchanger. A similar current was also the only current elicited by caged Ca2+ during the 5 msec latent period in wild type (WT) photoreceptors. To investigate possible modulatory effects of caged Ca2+ on the light-activated conductance, cells were first stimulated with a saturating light stimulus, itself incapable of releasing significant Ca2+, and then the photolytic flash was discharged during the response. During the rising phase of the response, photolysis of DM-nitrophen (but not nitr-5) induced a pronounced facilitation in WT photoreceptors. When photolysed during the plateau phase both DM-nitrophen and nitr-5 induced a rapid inactivation of the light-induced current. By contrast, in trp photoreceptors, which lack one class of Ca2+ permeable light-sensitive channel, photolysis of DM-nitrophen induced a significant facilitation during the falling phase of the response, but during the rising phase photolysis significantly depressed the overall response. In conclusion, caged Ca2+ failed to activate any channels in Drosophila photoreceptors but profoundly affected the light-dependent channels once they have been activated.

Acetates↗
PubMed1995

TRP, a protein essential for inositide-mediated Ca2+ influx is localized adjacent to the calcium stores in Drosophila photoreceptors.

The Drosophila transient receptor potential (trp) gene product (TRP) shows some structural similarity to vertebrate voltage-gated Ca2+ channels. It appears to function as a novel Ca2+ channel responsible for light stimulated, inositol trisphosphate (InsP3)-mediated Ca2+ entry in the fly retina. The subcellular localization of TRP protein was determined in this study using immunohistochemical staining with anti-TRP antibody (MAb83F6). TRP was localized to the base of the microvilli in a region adjacent to the presumed InsP3-sensitive Ca2+ stores. This specific localization was supported by measuring the magnitude of a TRP-dependent inward current that results from spontaneous activation of the light-sensitive channels during whole-cell recordings (the rundown current, RDC). We found that reduction of the microvilli area through genetic dissection with the opsin null mutant, ninaEora, was correlated with a pronounced enhancement of the TRP-dependent inward current relative to wild type, suggesting that the TRP-dependent current was not produced along the length of the microvilli. We suggest that the functional localization of the TRP protein is on the plasma membrane loop found along the base of the rhabdomeric microvillus. Thus, the TRP channel may function in concert with the InsP3-sensitive Ca2+ stores.

Animals↗
PubMed1994

Spontaneous activation of light-sensitive channels in Drosophila photoreceptors.

In Drosophila photoreceptors light induces phosphoinositide hydrolysis and activation of Ca(2+)-permeable plasma membrane channels, one class of which is believed to be encoded by the trp gene. We have investigated the properties of the light-sensitive channels under conditions where they are activated independently of the transduction cascade. Whole-cell voltage clamp recordings were made from photoreceptors in a preparation of dissociated Drosophila ommatidia. Within a few minutes of establishing the whole-cell configuration, there is a massive spontaneous activation of cation-permeable channels. When clamped near resting potential, this "rundown current" (RDC) accelerates over several seconds, peaks, and then relaxes to a steady-state which lasts indefinitely (many minutes). The RDC is invariably associated with a reduction in sensitivity to light by at least 100-fold. The RDC has a similar absolute magnitude, reversal potential, and voltage dependence to the light-induced current, suggesting that it is mediated by the same channels. The RDC is almost completely (> or = 98%) blocked by La3+ (10-20 microM) and is absent, or reduced and altered in the trp mutant (which lacks a La(3+)-sensitive light-dependent Ca2+ channel), suggesting that it is largely mediated by the trp-dependent channels. Power spectra of the steady-state noise in the RDC can be fitted by simple Lorentzian functions consistent with random channel openings. The variance/mean ratio of the RDC noise suggests the underlying events (channels) have conductances of approximately 1.5-4.5 pS in wild-type (WT), but 12-30 pS in trp photoreceptors. Nevertheless, the power spectra of RDC noise in WT and trp are indistinguishable, in both cases being fitted by the sum of two Lorentzians with a major time constant (effective "mean channel open time") of 1-2 ms and a minor component at higher frequencies (approximately 0.2 ms). This implies that the noise in the WT RDC may actually be dominated by non-trp-dependent channels and that the trp-dependent channels may be of even lower unit conductance.

Animals↗
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↗