Phospholipase C and termination of G-protein-mediated signalling in vivo.

In Drosophila photoreceptors, phospholipase C (PLC) and other signalling components form multiprotein structures through the PDZ scaffold protein INAD. Association between PLC and INAD is important for termination of responses to light; the underlying mechanism is, however, unclear. Here we report that the maintenance of large amounts of PLC in the signalling membranes by association with INAD facilitates response termination, and show that PLC functions as a GTPase-activating protein (GAP). The inactivation of the G protein by its target, the PLC, is crucial for reliable production of single-photon responses and for the high temporal and intensity resolution of the response to light.

A visual pigment with two physiologically active stable states.

Red illumination of a Balanus amphitrite photoreceptor that has been adapted to blue light leads to prolonged depolarization in the late receptor potential. This depolarization can be switched off by further exposure to a blue stimulus. The early receptor potential in this cell is purely depolarizing or largely hyperpolarizing; the former is true if the cell has been adapted to red light, and the latter, if blue light has been used. The color-adaptation "memories" for both early and late receptor potentials appear to be permanent. The existence of two stable states for the early receptor potential directly implies a pigment with two stable states, and these apparently contribute antagonistically to the late receptor potential.

Fluorescence of photoreceptor cells observed in vivo.

Most rhabdomeres in the eye of the fly (Musca domestica) are fluorescent. One kind of fluorescent emission emanates from a photoproduct of the visual pigment, other kinds may be ascribed to photostable pigments. These phenomena provide not only a means of spectrally mapping the retina but also a new spectroscopic tool for analyzing the primary visual processes in vivo.

Calmodulin regulation of calcium stores in phototransduction of Drosophila.

Phototransduction in Drosophila occurs through the ubiquitous phosphoinositide-mediated signal transduction system. Major unresolved questions in this pathway are the identity and role of the internal calcium stores in light excitation and the mechanism underlying regulation of Ca2+ release from internal stores. Treatment of Drosophila photoreceptors with ryanodine and caffeine disrupted the current induced by light, whereas subsequent application of calcium-calmodulin (Ca-CaM) rescued the inactivated photoresponse. In calcium-deprived wild-type Drosophila and in calmodulin-deficient transgenic flies, the current induced by light was disrupted by a specific inhibitor of Ca-CaM. Furthermore, inhibition of Ca-CaM revealed light-induced release of calcium from intracellular stores. It appears that functional ryanodine-sensitive stores are essential for the photoresponse. Moreover, calcium release from these stores appears to be a component of Drosophila phototransduction, and Ca-CaM regulates this process.

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.

The light response of Drosophila photoreceptors is accompanied by an increase in cellular calcium: effects of specific mutations.

Photoreceptors of dissociated Drosophila retinae were loaded with the fluorescent Ca2+ indicators, fluo-3 and Calcium Green-5N. In fluo-3-loaded, wild-type photoreceptors, a rapid increase in fluorescence (Ca2+ signal) accompanied the light-evoked inward current. Removal of extracellular Ca2+ greatly reduced the Ca2+ signal, indicating Ca2+ influx as its major cause. In Calcium Green-5N-loaded trp mutants, which lack a large fraction of the Ca2+ permeability underlying the light-evoked inward current, the Ca2+ signal was smaller relative to wild-type photoreceptors. Fluo-3-loaded norpA mutant photoreceptors, which lack a light-activated phospholipase C, generated no light-evoked inward current and no Ca2+ signal. The phosphoinositide pathway therefore appears necessary for both excitation and changes in cytosolic free Ca2+ concentration.

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.

The roles of trp and calcium in regulating photoreceptor function in Drosophila.

Invertebrate photoreceptors use the ubiquitous inositol-lipid signaling pathway for phototransduction. This pathway depends on Ca2+ release from internal stores and on Ca2+ entry via light-activated channels to replenish the loss of Ca2+ in those stores. The Drosophila transient receptor potential (TRP) protein is essential for the high Ca2+ permeability and other biophysical properties of these light-activated channels, which affect both excitation and adaptation in photoreceptor cells. Physiological and heterologous expression studies indicate that TRP is a putative subunit of a surface membrane channel that can be activated by depletion of internal Ca2+ stores. Furthermore, trp is an archetypal member of a multigene family whose products share a structure that is highly conserved throughout evolution, from worms to humans.

A common mechanism underlies vertebrate calcium signaling and Drosophila phototransduction.

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP(3)R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP(3) branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis we applied the membrane-permeant InsP(3)R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP(3)R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP(3)-mediated Ca(2+) release and store-operated Ca(2+) entry in Xenopus oocytes at a stage operating after production of InsP(3) but before the opening of the surface membrane Cl(-) channels by Ca(2+). We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP(3) branch of the inositol lipid-signaling pathway.

Novel mechanism of massive photoreceptor degeneration caused by mutations in the trp gene of Drosophila.

The Drosophila trp gene encodes a light-activated Ca(2+) channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant, Trp(P365), that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes, Trp(P365) is a trp allele because a Trp(P365) transgene induces the mutant phenotype in a wild-type background, and a wild-type trp transgene in a Trp(P365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp(P365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp(P365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

Light-induced reduction in excitation efficiency in the trp mutant of Drosophila.

In the transient receptor potential (trp) mutant of Drosophila, the receptor potential appears almost normal in response to a flash but quickly decays to baseline during prolonged illumination. Photometric and early receptor potential measurements of the pigment suggest that the pigment is normal and that the decay of the trp response during illumination does not arise from a reduction in the available photopigment molecules. However, there is reduction in pigment concentration with age. Light adaptation cannot account for the decay of the trp response during illumination: in normal Drosophila a dim background light shortens the latency and rise time of the response and also shifts the intensity response function (V-log I curve) to higher levels of light intensity with relatively little reduction in the maximal amplitude (Vmax) of response. In the trp mutant, a dim background light or short, strong adapting light paradoxically lengthens the latency and rise time of the response and substantially reduces Vmax without a pronounced shift of the V-log I curve along the I axis. The effect of adapting light on the latency and V-log I curve seen in trp are associated with a reduction in effective stimulus intensity (reduction in excitation efficiency) rather than with light adaptation. Removing extracellular Ca+2 reduces light adaptation in normal Drosophila, as evidenced by the appearance of "square" responses to strong illumination. In the trp mutant, removing extracellular Ca+2 does not prevent the decay of the response during illumination.

Microspectrophotometric evidence for two photointerconvertible states of visual pigment in the barnacle lateral eye.

Microspectrophotometrically derived difference spectra from the barnacles Balanus amphitrite and B. eburneus show that a blue illumination after an orange illumination causes a decrease in absorption in the blue region and an increase in absorption in the green-yellow region, with an isosbestic point around 535 nm. Orange-following-blue illumination causes the reverse changes. The dark time between the adapting and measuring lights has no influence on the data. The results confirm previously reported ERP measurements which indicate that the barnacle visual pigment has two photointerconvertible dark-stable states. If one assumes a Dartnall nomogram shape for the two absorption spectra, a best fit to the observed difference spectra is obtained with nomograms peaking at 492 nm and 532 nm, with a peak absorbance ratio around 1.6:1. These two nomograms fit very well the ERP action spectra of metarhodopsin and rhodopsin, respectively, thus indicating that the ERP is a reliable measure of visual-pigment changes in the barnacle. The existence of a photostable blue pigment is demonstrated in B. eburneus and in some of B. amphitrite receptors, and the possible influence of this photostable pigment on the various action spectra measured in the barnacle is discussed.

The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin.

Most of the photoreceptors of the fly compound eye have high sensitivity in the ultraviolet (UV) as well as in the visible spectral range. This UV sensitivity arises from a photostable pigment that acts as a sensitizer for rhodopsin. Because the sensitizing pigment cannot be bleached, the classical determination of the photosensitivity spectrum from measurements of the difference spectrum of the pigment cannot be applied. We therefore used a new method to determine the photosensitivity spectra of rhodopsin and metarhodopsin in the UV spectral range. The method is based on the fact that the invertebrate visual pigment is a bistable one, in which rhodopsin and metarhodopsin are photointerconvertible. The pigment changes were measured by a fast electrical potential, called the M potential, which arises from activation of metarhodopsin. We first established the use of the M potential as a reliable measure of the visual pigment changes in the fly. We then calculated the photosensitivity spectrum of rhodopsin and metarhodopsin by using two kinds of experimentally measured spectra: the relaxation and the photoequilibrium spectra. The relaxation spectrum represents the wavelength dependence of the rate of approach of the pigment molecules to photoequilibrium. This spectrum is the weighted sum of the photosensitivity spectra of rhodopsin and metarhodopsin. The photoequilibrium spectrum measures the fraction of metarhodopsin (or rhodopsin) in photoequilibrium which is reached in the steady state for application of various wavelengths of light. By using this method we found that, although the photosensitivity spectra of rhodopsin and metarhodopsin are very different in the visible, they show strict coincidence in the UV region. This observation indicates that the photostable pigment acts as a sensitizer for both rhodopsin as well as metarhodopsin.

Fast electrical potentials arising from activation of metarhodopsin in the fly.

The cellular origin and properties of fast electrical potentials arising from activation of Calliphora photopigment were investigated. It was found by intracellular recordings that only the corneal-negative M1 phase of fly M potential arises in the photoreceptors' membrane. This M1 phase has all the accepted characteristics of an early receptor potential (ERP). It has no detectable latency, it survives fixation with glutaraldehyde, it is linear with light intensity below pigment saturation, and it is linear with the amount of metarhodopsin activated by light. The Calliphora ERP was found, however, to be exceptional because activation of rhodopsin, which causes the formation of metarhodopsin in 125 microsecond (25 degrees C), was not manifested in the ERP. Also, the extracellularly recorded ERP was not proportional to the rate of photopigment conversion. The corneal-positive M2 phase of the M potential was found to arise from second-order lamina neurons (L neurons). Intracellular recordings from these cells showed a fast hyperpolarizing potential, which preceded the normal hyperpolarizing transient of these cells. This fast potential appeared only when metarhodopsin was activated by a strong flash. The data indicate that the intracellularly recorded positive ERP, which arises from activation of metarhodoposin, elicits a hyperpolarizing fast potential in the second-order neuron. This potential is most likely the source of the corneal-positive M potential.

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.

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.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process.

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. II. Antagonistic interactions.

In the preceding article, we investigated the spatial properties of the induction of the prolonged depolarizing afterpotential (PDA) by shifting visual pigment from the rhodopsin (R) to the metarhodopsin (M) state in the barnacle photoreceptor. In this work, we have studied the ranges within the cell of the antagonistic effects on the PDA of M-to-R transfer. When this transfer occurs during a PDA, it depresses the PDA; when it precedes PDA induction, it impedes that induction ("anti-PDA"). These ranges were previously shown (by a statistical technique) to be at least a few tens of nanometers within a half-second (D greater than 10(-13) cm2 s-1). We now demonstrate, with local illumination techniques in which a PDA was induced in one side of the cell and PDA depression or anti-PDA was induced in the other side, that both ranges are much smaller than the cell diameter (approximately 100 microns) within 30 s (D less than 10(-6)). We further show, using a less direct but shorter-range technique involving colored polarized light, that the interaction of the PDA with the anti-PDA is restricted to less than approximately 6 microns (D less than 6 X 10(-9)). This figure is quite low and suggests that the interaction may be confined to the pigment molecules, possibly in a complex of the type suggested in the preceding article.

Antagonistic components of the late receptor potential in the barnacle photoreceptor arising from different stages of the pigment process.

The late receptor potential (LRP) recorded in barnacle photoreceptor cells exhibits, at high light levels, a strong dependence on the color of the stimulus and of the preceding adaptation. Most strikingly, red illumination of a cell previously adapted to blue light results in a depolarization which may last for up to 30 min after the light goes off, while blue illumination of a cell previously adapted to red light cuts short this extended depolarization or prevents its induction by a closely following red light. Comparison of the action spectra for the stimulus-coincident LRP and for the extended depolarization and its curtailment with those previously measured for the early receptor potential (ERP) confirms that these phenomena derive from the same bi-stable pigment as the ERP. The stimulus-coincident response and the extended depolarization appear to arise from substantial activation of the stable 532 nm state of the pigment, while activation of the stable 495 state depresses or prevents the extended depolarization and probably also depresses the stimulus-coincident response. Since either process can precede the other, with mutually antagonistic effects, one is not simply the reversal of the other; they must be based on separate mechanisms. Furthermore, comparison with ERP kinetics shows that both processes involve mechanisms additional to the pigment changes, as seen in the ERP. A model is proposed and discussed for the LRP phenomena and their dependences on wavelength, intensity, and duration of illumination based on excitor-inhibitor interactions.

Early receptor potential evidence for the existence of two thermally stable states in the barnacle visual pigment.

The early receptor potential (ERP) in the barnacle photoreceptor is shown by intracellular recording to exhibit a strong dependence on the color of the stimulus and of the preceding adaptation. The adaptation effects appear to be stable for at least 3 h in the dark. Most strikingly, the ERP is positive after red adaptation and mainly negative after blue adaptation. The simplest hypothesis which accounts for these observations is that two thermally stable pigment states with different absorption spectra contribute to the ERP. All ERP responses appear to be consistent with the sums of different ratios of the ERP's of the two pure states. The relative populations of the two states are shown to vary reciprocally, suggesting that the two are states of the same closed pigment cycle. Both states have approximately Dartnall nomogram-shaped absorption spectra, one peaked near 495 nm, and the other near 532 nm.