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Journal of Physiology (2002), 543.2, pp. 481-494
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.022772

Role of protein kinase C in light adaptation of molluscan microvillar photoreceptors

Giuseppe Piccoli*, Maria del Pilar Gomez*† and Enrico Nasi*†

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

The mechanisms by which Ca²⁺ regulates light adaptation in microvillar photoreceptors remain poorly understood. Protein kinase C (PKC) is a likely candidate, both because some sub-types are activated by Ca²⁺ and because of its association with the macromolecular 'light-transduction complex' in Drosophila. We investigated the possible role of PKC in the modulation of the light response in molluscan photoreceptors. Western blot analysis with isoform-specific antibodies revealed the presence of PKC in retinal homogenates. Immunocytochemistry in isolated cell preparations confirmed PKC localization in microvillar photoreceptors, preferentially confined to the light-sensing lobe. Light stimulation induced translocation of PKC immunofluorescence to the photosensitive membrane, an effect that provides independent evidence for PKC activation by illumination; a similar outcome was observed after incubation with the phorbol ester PMA. Several chemically distinct activators of PKC, such as phorbol-12-myristate-13-acetate (PMA), (-)indolactam V and 1,2,-dioctanoyl-sn-glycerol (DOG) inhibited the light response of voltage-clamped microvillar photoreceptors, but were ineffective in ciliary photoreceptors, in which light does not activate the G_q/PLC cascade, nor elevates intracellular Ca²⁺. Pharmacological inhibition of PKC antagonized the desensitization produced by adapting lights and also caused a small, but consistent enhancement of basal sensitivity. These results strongly support the involvement of PKC activation in the light-dependent regulation of response sensitivity. However, unlike adapting background light or elevation of [Ca²⁺]_i, PKC activators did not speed up the photoresponse, nor did PKC inhibitors antagonize the accelerating effects of background adaptation, suggesting that modulation of photoresponse time course may involve a separate Ca²⁺-dependent signal.

(Received 17 April 2002; accepted after revision 21 June 2002)
Corresponding author E. Nasi: Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA. Email: enasi{at}bu.edu

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

In photoreceptor cells the ability to modulate sensitivity in response to changes in ambient illumination is crucial to expand the operating range and prevent response saturation. In vertebrate rods and cones, Ca²⁺ is a critical mediator of the light adaptation process (reviewed by Pugh et al. 1999) and many of its target effectors are known: for example, Ca²⁺ modulates (via the Ca²⁺-binding proteins GCAPs) guanylate cyclase activity (Koch & Stryer, 1988), the lifetimes of activated rhodopsin and the consequent stimulation of phosphodiesterase (PDE) (via recoverin; Kawamura, 1993), the affinity of the channels for cGMP (via calmodulin; Hsu & Molday, 1993), and the catalytic activity of rhodopsin (Lagnado & Baylor, 1994). These coordinated functions decrease sensitivity and accelerate dark-current recovery during background illumination. In rhabdomeric photoreceptors Ca²⁺ has also long been postulated to play a crucial role in light adaptation, since the seminal demonstration in Limulus, and later in Apis mellifera, that Ca²⁺ injection desensitizes the photoresponse, whereas EGTA retards the decay of the light response during sustained illumination (Lisman & Brown, 1972, 1975; Bader et al. 1976). The mechanisms by which Ca²⁺ contributes to light adaptation in invertebrate microvillar photoreceptors remain poorly understood, but protein kinase C (PKC) has emerged as a likely candidate: in Drosophila, PKC has been shown to be associated with the macromolecular transduction complex coordinated by the scaffolding protein InaD (Tsunoda et al. 1997; Adamski et al. 1998; Xu et al. 1998). Morevover, InaC, a Drosophila mutant lacking an eye-specific PKC (Ranganathan et al. 1991), has been reported by Hardie et al. (1993) to be defective in light adaptation (but see Smith et al. 1991 for an alternative interpretation). Experimental evidence linking PKC manipulations to changes in the photoresponse in wild-type photoreceptors has recently begun to emerge: in Lima, activators of PKC have been shown to cause an increase in membrane conductance, but, in addition, they also induce a conspicuous loss in light sensitivity (Gomez & Nasi, 1998); desensitization of the light response by these agents was subsequently reported also in Limulus, and their target was localized upstream in the transduction cascade, possibly the phospholipase C (PLC) (Dabdoub & Payne, 1999). The present report systematically examined the conjecture that PKC may be an effector in the light adaptation process of molluscan photoreceptors. We obtained further supporting evidence for the specificity of the PLC pathway involvement in the desensitization caused by diacylglycerol (DAG) surrogates; additionally, we employed immunodetection methods to determine the presence and sub-type identity of PKCs in Lima eyes, and to establish the distinct localization of a Ca²⁺-sensitive isoform in the light-transducing lobe of rhabdomeric photoreceptors. Examination of the changes in PKC spatial distribution under different experimental conditions provided independent evidence for its activation by DAG analogues and by adapting lights, an important clue to establish that PKC stimulation is causally related to the loss of sensitivity caused by background illumination and conditioning stimuli. Finally, we demonstrated that pharmacological inhibition of PKC interferes with the normal process of light adaptation. Aspects of this report were previously presented in preliminary form (Piccoli et al. 2001).

	METHODS

Top Abstract Introduction Methods Results Discussion References

Cell dissociation procedures

Complete eyecups of Lima scabra (Carolina Biological, Burlington, NC, USA) were dissected under dim red light ( > 650 nm), incubated in 0.6 % collagenase (Worthington Type II) and 0.4 % trypsin (Sigma Type III) for 40 min at 24 °C, washed in 3 % fetal calf serum, and gently triturated with a fire-polished Pasteur pipette, as previously described (Nasi, 1991a). Retinas of Pecten irradians, obtained from the Marine Resources Center at the Marine Biological Laboratory (Woods Hole, MA, USA), were incubated in pronase (Boehringer, 12 ml ml^-1) for 40-50 min at 22 °C and treated as above (Nasi & Gomez, 1992; Gomez & Nasi, 1994). After plating, the recording flow-chamber was continuously superfused with artificial sea water (ASW) containing (mM): 480 NaCl, 10 KCl, 49 MgCl₂, 10 CaCl₂, 10 Hepes and 5 glucose, pH 7.8 (NaOH). Isolated photoreceptor cells remained viable for 4-6 h.

Whole-cell recording

Whole-cell patch pipettes were fabricated from borosilicate glass (Garner Glass 7052), fire-polished, and filled with an intracellular solution containing (mM): 100 KCl, 200 potassium aspartate or potassium glutamate, 5 MgCl₂, 5 Na₂ATP, 12 NaCl, 1 EGTA, 300 sucrose, 10 Hepes, 0.2 GTP, pH 7.3 (KOH). For elevated Ca²⁺ (1 µM) we used a suitable mixture of CaCl₂ with either EGTA (as determined by the program Chelator), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEEDTA), which is a better buffer at the desired target concentration of calcium; photocurrents were similar with either solution. Electrode resistance measured in ASW was 2-4 M; series resistance was routinely compensated (maximum residual error < 2 mV). All recordings were made at room temperature (20-22 °C).

Chemical stimulation

For extracellular chemical stimulation a puffer pipette (tip diameter ~2-3 µm) was lowered automatically to a pre-set position near the target cell (~30-50 µm) by a programmable positioner (Eppendorf, Hamburg, Germany). Test solutions were ejected by applying pressurized nitrogen, under the control of a solenoid-activated valve. The efficacy of the local perfusion was assessed in test trials in which the long-wavelength fluorescent dye Cy5.18 (50-100 µM; BDS, Pittsburgh, PA, USA) was added to the puffer pipette solution. The ejection plume, visualized by means of an intensified camera (EEV, Chelmsford, UK), entirely engulfed the target cell, and the time constant of solution change at that position, determined with a photomultiplier that measured fluorescence in a 5 µm 5 µm optical window, was ~100 ms (Gomez & Nasi, 1996). Substances to be dialysed intracellularly were added to the patch pipette solution from freshly made stocks. With small molecules, we have previously shown that the cytosol in the microvillar lobe fully equilibrates with the internal solution (Gomez & Nasi, 1996), and the process occurs within 2-3 min.

The water-insoluble DAG surrogates phorbol-12-myristate-13-acetate (PMA; Calbiochem, San Diego, CA, USA) (-)indolactam V (LC Laboratories, Woburn, MA, USA) and 1,2-dioctanoyl-sn-glycerol (DOG; Sigma, St Louis, MO, USA and Calbiochem) were dissolved in DMSO, and the stock solutions were aliquoted and stored under argon at -80 °C. The final concentration of DMSO present either in the internal solution or in the puffer pipette solution was < 0.5 %. Control experiments demonstrated that concentrations of this solvent up to 5 % were inert on the membrane current and on the light response (Gomez & Nasi, 1998). The PKC inhibitors Ro-32-0432, chelerythrine, and the peptide PKC(19-36) were obtained from Calbiochem.

Light stimulation

A dual optical stimulator was used to deliver test flashes and adapting backgrounds. The first beam, from a 100 W tungsten- halogen light source (Oriel, Stratford, CT, USA), was passed through a heat-absorbing filter (>95 % rejection for > 800 nm). A solenoid-driven shutter (Uniblitz; Vincent Associates, Rochester, NY, USA) and calibrated neutral density filters were used to control duration and intensity of stimulation. A pin-hole and a field lens restricted the illuminated region to a focused spot on the chamber (~150 µm). A beam-splitter above the microscope condenser combined this beam with that of the microscope illuminator. The second stimulator consisted of a 100 W mercury arc lamp (Zeiss), a cold mirror ( < 690 nm) a shutter, and a light guide coupled to the epifluorescence port of the microscope. Light was reflected by a dichroic mirror ( < 650 nm) and focused onto the preparation by the microscope objective. The intensity of light stimuli is expressed as log₁₀ (I/I_o), where I_o, the reference light, was 9.3 10^-3 W cm^-2, as measured with a radiometer (UDT, Hawthorne, CA, USA). Calibrated neutral-density filters (Melles Griot, Irvine, CA, USA) provided controlled attenuation. During experimental manipulations the cells were illuminated with near-IR light using a long-pass filter ( > 780 nm; Andover Corporation, Salem, NH, USA) and viewed with the aid of a TV camera (Panasonic, Secaucus, NJ, USA). The infrared illuminator was turned off for several minutes before testing light responses.

Intracellular Ca²⁺ monitoring

To measure Ca²⁺ fluorescence, the excitation light from a 75 W xenon arc source (PTI, South Brunswick, NJ, USA) was reflected off a cold mirror (_c = 670 nm; Omega, Brattleboro, VT, USA), passed through an interference filter (_max = 480 nm, 40 nm band width; Chroma, Brattleboro, VT, USA) and fed to the epi-illumination port of the inverted microscope via a liquid light-guide (Oriel); the filter block in the turret contained a 505 nm dichroic reflector, and a barrier filter (_max of 535 nm, 50 nm band width; Chroma). At the camera port, an adjustable, positionable mask (Nikon) was used to circumscribe the collected fluorescence to a defined rectangular region. The output light was further split by an additional dichroic mirror (_c = 610 nm), which diverted the fluorescence signal to a photomultiplier tube (PMT; Hammamatsu, Bridgewater, NJ, USA) connected to a preamplifier-discriminator and a photon counter (Advanced Research Instrumentation, Boulder, CO, USA); long wavelength light was directed to the camera used to visualize the cells. Electromechanical shutters (Vincent Associates) controlled excitation light and PMT exposure. Photoreceptors were loaded with the fluorescent Ca²⁺ indicators Fluo-4 or Calcium Green 5N (30-85 µM; Molecular Probes, Eugene, OR, USA) via the patch pipette; before testing, ~4-6 min of internal dialysis time were allowed to elapse.

Immunodetection

For immunocytochemistry, enzymatically dispersed retinal cell suspensions were plated onto coverslips pre-treated with collagen and concanavalin A. Cells were fixed in ASW containing 2 % paraformaldehyde and 0.075 % picric acid for 15 min, washed in 0.1 M phosphate buffer (PBS), and permeabilized in 0.2 % Triton-X for 5 min on ice. Subsequently, the coverslips were immersed in PBS with 1 % goat serum for 10 min. Incubation with primary antibodies (1:1000) in PBS + 0.5 % BSA (40 min) was followed by two washes in PBS and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibodies (Abs; 1:120, 30 min). After the final washes (three times in PBS) the coverslips were mounted onto glass slides using 50 % glycerol, and sealed with nail polish. Cells were subsequently visualized in a conventional fluorescence microscope (Zeiss Axioplan) equipped with a digital camera (Zeiss Axiocam), using a 546 nm (bandpass) excitation filter, a 580 nm dichroic reflector and a 590 nm long-pass emission filter. Fluorescence distribution was analysed by constructing line intensity profiles from background-subtracted image files, using the NIH/Scion Image program.

For Western blots, retinas were homogenized (teflon/glass) in the presence of protease inhibitors (100 µM phenylmethylsulfonyl fluoride (PMSF), 1 µM pepstatin and 0.1 % Sigma protease inhibitor cocktail), acetone-precipitated for 1 h at -20 °C, and centrifuged 20 min at 10 000 g. The pellet was air-dried, resuspended in sample buffer and separated by SDS-PAGE (7 %). Proteins were electrotransferred (1 h, 100 V) onto a nitrocellulose membrane which was blocked overnight with 3 % bovine serum albumin (BSA). The membrane was then sequentially incubated with primary antibodies (1:2000, 45 min), washed in Tris buffered saline (TBS) and incubated in alkaline phosphatase-conjugated anti-rabbit secondary Abs (1:1000, 2 h). In order to simultaneously screen antibodies against several PKC isoforms, the gel consisted of a single, wide preparative lane (plus a narrow lane for standards) and, after electro-transferring the proteins, incubation in the primary Abs was carried out in a multichannel device (Surf Blot, Idea Scientific Co., Corvallis, OR, USA), which allows many separate canals to be independently perfused. After the final washes, the nitrocellulose membrane was developed in Western Blue (Promega, Madison, WI, USA).

Pan-specific anti-PKC antibodies (rabbit) were from Chemicon (Temecula, CA, USA; Cat. AB-1610). Isoform-specific, affinity purified rabbit anti-PKC polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA; Cat. sc-208). These antibodies were raised against a peptide that is completely conserved in human, rat and mouse, and maps at the carboxy terminus of PKC; the same company supplied the corresponding blocking peptide. The antiserum against a C-terminus peptide of Aplysia Ca²⁺-dependent PKC, Apl I, was a generous gift from Dr Wayne Sossin (McGill University, Montreal, Canada). Anti-rabbit TRITC-conjugated secondary antibodies were from Sigma, whereas alkaline phosphatase-conjugated anti-rabbit secondary antibodies were purchased from Promega.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Figure 1 summarizes the most salient arguments in support of a key role for Ca²⁺ in light adaptation, as documented in the preparation used in the present study, namely, isolated Lima rhabdomeric photoreceptors. Panel A (left) illustrates a typical response elicited by a brief test flash initially under dark-adapted conditions and subsequently superimposed upon a sustained light; background illumination considerably reduced the amplitude of the flash response and accelerated its kinetics. The trace on the right shows that a similar effect can be obtained in a dark-adapted photoreceptor, by perfusing the cytosol with a solution containing elevated (1 µM) buffered Ca²⁺ (see Methods), instead of the standard intracellular solution (i.e. 1 mM EGTA, no added Ca²⁺). Panel B shows that photostimulation rapidly increases Ca²⁺ concentration within the light-transducing lobe. Whole-cell membrane current and fluorescence from the rhabdomere were measured simultaneously in photoreceptors loaded with the Ca²⁺ indicator Calcium Green 5N. Light stimulation invariably caused a large, rapid increase of cytosolic Ca²⁺ (left; n > 50); the light-induced Ca²⁺ rise survived removal of extracellular Ca²⁺ (right), indicating that, as in Limulus (Brown & Blinks, 1974), it is primarily due to release from internal stores (n = 9; see also Gomez & Nasi, 1998). Finally, panel C demonstrates that the decay of the photocurrent during sustained illumination (a feature that is at least partly attributable to the development of light adaptation) is retarded by chelation of intracellular Ca²⁺. Light steps were delivered to dark-adapted photoreceptors dialysed with standard intracellular solution (left), or with a solution containing a higher concentration of EGTA (5 mM, right). In control conditions the photocurrent rapidly returned to the baseline during the sustained illumination, as previously described (Nasi, 1991b); a similar behaviour has been reported in other molluscan photoreceptors, such as those of Aplysia (Jacklet, 1969). Because the flash response accelerates and its sensitivity curve shifts to the right as a function of background illumination, the pronounced decay with sustained light seems to reflect strong susceptibility to adaptation, rather than 'exhaustion' of excitation, as postulated for the phenotype of the Drosophila trp mutant (Minke, 1982). Upon increasing the Ca²⁺-buffering capacity of the cell two effects occurred: in the first place, the rising phase of the response became more sluggish, a phenomenon extensively documented in other species (Lisman & Brown, 1975; Shin et al. 1993; Hardie, 1995a) and thought to reflect the reduction of the early facilitatory influence of Ca²⁺ on transduction. More pertinent to the present topic, the photocurrent exhibited a much reduced fall during the stimulus, as first described in Limulus (Lisman & Brown, 1975), so that a significant fraction of the light-evoked current remained at the end of the 2 s illumination period (n = 4). In summary, Lima microvillar photoreceptors exhibit the hallmarks of Ca²⁺-dependent sensory adaptation described in other standard preparations: an elevation of intracellular Ca²⁺ accompanies photoexcitation, and appears to be both necessary and sufficient for light-induced desensitization.

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Figure 1. Ca2+ as a mediator of light adaptation in Lima microvillar photoreceptors A, elevated Ca2+ mimics light adaptation: comparison of the photocurrent evoked by a 100 ms flash (-3.8 log) in dark-adapted conditions and in the presence of continuous background illumination (-4.8 log). The adapting light desensitized the response to the test flash and accelerated its kinetics. The two traces were vertically offset for clarity. Internal perfusion with a solution containing 1 µM Ca2+ (right) in another cell maintained in dark-adapted conditions produced effects that resemble those of light adaptation (flash intensity -0.6 log). B, illumination increases cytosolic Ca2+ in the light-sensitive lobe. The photoreceptors were loaded with the Ca2+ indicator Calcium Green 5N via the patch pipette (75 and 38 µM, respectively) and the fluorescence emission within a small window (~5 µm 5 µm) encompassing only the rhabdomeric lobe (lower traces) was measured simultaneously with the whole-cell current (top traces). After ~5 min of internal dialysis, the epi-illumination beam was turned on for 500 ms: the abrupt jump upon opening the shutter reflects the basal fluorescence (dotted lines), after which the optical signal climbed steeply, indicating a large increase in cytosolic Ca2+ coincident with the photocurrent. The traces on the right were obtained in a cell superfused for ~8 min with Ca2+-free solution. The large photoinduced Ca2+ transient was not eliminated by removal of extracellular Ca2+, indicating that it is due primarily to internal release. C, Ca2+ chelators slow down the decline of the light response during illumination. Comparison of the photocurrent evoked by 1 s steps of light in two photoreceptors, one dialysed with standard intracellular solution (1 mM EGTA, left), and the other with 5 mM EGTA (right). Whereas in control conditions the photocurrent rapidly returned to baseline during the stimulus (a manifestation of adaptation), increasing the Ca2+-buffering capacity of the cell retarded the decline of the light response, and at the termination of the stimulus a sizeable plateau remained. Relative light intensity -2.2 log and 0.6 log, respectively; holding potential (Vh) -50 mV in all recordings.

DAG analogues were previously shown to desensitize the light response (Gomez & Nasi, 1998; Dabdoub & Payne, 1999). To further document the specificity of such effects, we compared the influence of these agents on microvillar vs. ciliary photoreceptors (which co-exist with rhabdomeric cells in the double retina of several marine molluscs; reviewed by McReynolds, 1976). The latter cell type utilizes a fundamentally different transduction cascade that relies on mobilization of cGMP (Gomez & Nasi, 1995, 2000) without involving the phospholipase C signalling pathway (and thus no light-induced production of DAG; Gomez & Nasi, 1995), nor any elevation of cytosolic Ca²⁺ (Gomez & Nasi, 1999). Panel A of Fig. 2 illustrates the dramatic reduction in light responsiveness in Lima microvillar photoreceptors, produced by local application of three chemically unrelated activators of PKC, known to bind to the same site targeted by DAG (Sugimura, 1982; Sharkey et al. 1984). Photocurrents were recorded in control conditions (lower traces) and after exposure to the phorbol ester PMA (1 µM, left), the alkaloid (-)indolactam V (50 µM, middle) or the DAG derivative DOG (100 µM, right); n > 20 in each of the conditions. The loss of sensitivity was > 1.5 log, as previously demonstrated, and the effects were stereospecific (Gomez & Nasi, 1998; Dabdoub & Payne, 1999). A dramatic inhibition of the light response by the same three agents was also observed in microvillar photoreceptors isolated from the related species Pecten irradians (not shown; n = 4, 1 and 2, respectively). DAG analogues were previously shown to exert stimulatory effects on these photoreceptors (Gomez & Nasi, 1998); however, the inward currents elicited chemically never exceeded 1 nA, far less than the extent of photocurrent reduction, indicating that the desensitization is not simply a result of a direct occlusive interaction between DAG analogues and light for channel activation. Figure 2B shows that, by contrast, in a Lima ciliary (hyperpolarizing) photoreceptor, the outward photocurrent was completely impervious to local application of 100 µM DOG. Similarly, in Pecten ciliary photoreceptors exposure to 100 µM (-)indolactam V was ineffective (n = 3). The lack of effect of DAG analogues on visual cells that do not utilize the G_q/PLC signalling cascade strengthens the suggestion that the pharmacologically-induced desensitization in microvillar photoreceptors is indeed mediated by activation of PKC.

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Figure 2. Specificity of the desensitizing effects of PKC activators A, light-evoked membrane current measured in different Lima microvillar photoreceptors, before (lower traces) and during application of various PKC activators by a puffer pipette positioned ~50 µm from the cell. Left: PMA (1 µM); middle: (-)indolactam V (50 µM,); right: DOG (100 µM). In all cases, the amplitude of the photocurrent was substantially depressed by the DAG surrogates. Traces were offset vertically for clarity. Light intensity was kept constant for each cell (log(I/Io) = 0, 0 and -0.9, respectively) and the holding potential was -50 mV. B, lack of effect of PKC activators on ciliary, invertebrate photoreceptors. Left: application of 100 µM DOG failed to alter the light-evoked outward current of a hyperpolarizing (ciliary) Lima photoreceptor (light intensity -2.1 log). Right: similarly, puffing 100 µM (-)indolactam V onto a Pecten ciliary cell produced no effect on the photoresponse (light intensity -1 log). Photoreceptors were voltage-clamped at -30 mV, near their resting potential.

Immunodetection, isoform identification and cellular localization of PKC

We used immunoreagents to seek additional clues in support of the notion that PKC may be involved in the regulation of the photoresponse by light. We first carried out Western blot analysis of Lima eye homogenates using a pan-specific antibody against conventional PKCs. A strong, if somewhat diffuse, band of approximately the appropriate molecular weight was detected (not shown). We then screened a battery of isoform-specific antibodies to clarify the identity of the particular subtype(s) present. Figure 3 (left) shows that at a dilution of 1:2000 there was a strong immunoreactivity only for the isoform. Upon increasing the concentration of the primary antibodies fourfold and extending the incubation time, very faint signals could also be seen with antibodies against the conventional sub-type I, and the novel PKCs and (not shown). An additional antibody raised against Aplysia PKC, Apl I, which is Ca²⁺-dependent (Sossin et al. 1996), was also strongly reactive (Fig. 3, right). In all the positive cases, the antibodies only detected a single band of similar molecular size, (~85 kDa), compatible with the reported molecular mass of most PKCs.

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Figure 3. Western blot detection of PKC in Lima eyes Proteins from eye homogenates were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes. Left: screening of a battery of isoform-specific anti-mammalian PKC polyclonal antibodies, detected with alkaline phosphatase-conjugated secondary antibodies. Strong immunoreactivity was only observed at a single band of ~85 kDa with antibodies against PKC. Right: another antibody against the C-terminal sequence of an Aplysia Ca2+-dependent PKC (Apl I) was also positive.

To assess PKC localization at the cellular level, we utilized the two positive antibodies raised against Apl I and PKC, respectively, and performed immuno-cytochemistry on enzymatically isolated retinal cells dark-adapted prior to fixation, as shown in Fig. 4. The top panels (A) show a bright-field micrograph (left) of control Lima rhabdomeric photoreceptors and its corresponding epi-fluorescence image (right). The cells were only incubated with TRITC-conjugated secondary antibodies, and the only visible signal arises from the strongly autofluorescent screening pigments localized in the intermediate region of the photoreceptors and in support cells. In fact, a similar pattern of fluorescence was obtained when the fluorescent antibodies were also omitted (not shown). The bottom panels (B) show fluorescence micrographs of cells that were incubated with anti-Aplysia PKC polyclonal antibodies. Strong immunofluorescence is visible both in the rhabdomeric lobe (R) and in the soma (S). Antibodies against mammalian PKC also strongly labelled microvillar photoreceptors, but in this case the pattern of staining was markedly different. Figure 5A shows that the signal was distinctly more concentrated in the rhabdomeric lobe, and in most cases the label was virtually undetectable in the somatic region. Within the rhabdomere, anti-PKC immunofluorescence was distributed throughout the cytoplasm, but tended to be more pronounced around the edges, suggesting some preferential accumulation near the membrane. Control slides in which the primary antibodies were either omitted (Fig. 5B) or pre-incubated with the blocking peptide (Fig. 5C) failed to reveal any signal in the R-lobe, and only the autofluorescence from screening pigment and support cells remained visible. The results therefore argue in favour of the presence of an -like isoform of PKC in molluscan microvillar photoreceptors, primarily confined to the light-transducing region. Because this pattern of localization, unlike that of Apl I, is suggestive of a specific involvement in some light-related process, subsequent experiments focused on the PKC isozyme.

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Figure 4. Antibodies against Aplysia PKC label enzymatically dissociated Lima microvillar photoreceptors A, bright field (left) and fluorescence (right) micrographs of control cells. After plating and fixation in the dark, the cells were incubated only with TRITC-conjugated antibodies, without prior exposure to the primary antibodies. The only detectable emission is the autofluorescence of the dark screening pigments, which are found in the middle region of the photoreceptors, and also in support cells (sc). R, rhabdomeric lobe; S, soma; calibration bar: 10 µm. B, fluorescence images of photoreceptors that were incubated with antibodies against Apl I, followed by TRITC-conjugated secondaries. PKC fluorescence is clearly visible both in the rhabdomeric lobe and in the soma, where its intensity is particularly pronounced.

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Figure 5. Selective immunolabelling of the light-transducing lobe by anti-PKC antibodies Lima eyes were dissociated and plated in the dark prior to fixation. A, PKC immunofluorescence appears largely confined to the rhabdomeric lobe (R) of a photoreceptor, and is nearly undetectable in the cell body (S; sc, support cell). Omission of the incubation with the primary antibodies (B), or pre-adsorption of the primary antibodies with the blocking peptide (C) eliminated the fluorescence from the microvillar lobe. Calibration bar: 10 µm.

Activation of PKC by chemical stimulation and by light

Activation of Ca²⁺- and DAG-dependent PKCs involves translocation from the cytosol to the membrane (Kraft et al. 1982). We compared the spatial distribution of PKC immunofluorescence under resting conditions and after exposure to phorbol-12-myristate-13-acetate (PMA), one of the most powerful activators of conventional and novel PKCs. To this end, dissociated photoreceptors plated onto coverslips were dark-adapted for 1 h, and then either directly fixed in paraformaldehyde, or superfused with PMA before fixation. All manipulations were done under near-IR light, using infrared viewers. The PMA concentration (1 µM) and the incubation time (3 min) were chosen to match the parameters that proved effective in depressing the photocurrent in electrophysiological experiments (see Fig. 2A and Gomez & Nasi, 1998). Cells were subsequently treated with anti PKC antibodies and processed as described above. Figure 6A shows fluorescence micrographs obtained with a high numerical aperture oil-immersion objective (n = 1.4, 63 ) to reduce the depth of field, so that the image approaches an optical section through the cell. In the PMA-treated photoreceptor (bottom, left) the fluorescence appears more sharply localized in the proximity of the rhabdomeric membrane, whereas little signal is visible in the cytosol, unlike in the control cell (top, left). To quantify the extent of the PMA-induced redistribution of PKC, the fluorescence intensity distribution was measured along a line emanating approximately from the centre of the rhabdomere (as drawn in the figure); the resulting profiles are plotted on the right of each micrograph. For each cell, the ratio of the fluorescence near the centre of the rhabdomeric lobe (F_cyt) vs. the edge (F_memb) was determined. The bar graph in Fig. 6B shows the average F_cyt/F_memb ratio obtained from 17 control cells and from 20 cells exposed to PMA. A Student's t test revealed that the difference between the two groups was significant at the 0.01 level. Additional slides were exposed to the inert PMA isomer 4-PMA (1 µM) in the dark, and the fluorescence profiles failed to differ significantly from control cells.

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Figure 6. Translocation of PKC induced by phorbol ester A, isolated photoreceptors were plated and dark-adapted for 1 h, and subsequently either directly fixed in paraformaldehyde (upper panels), or dipped in ASW containing 1 µM PMA for 3 min and then fixed (lower panels). Immunocytochemistry with anti-PKC antibodies revealed a different spatial pattern of immunofluorescence in the two conditions: whereas in control cells a significant fraction of PKC seems to be distributed diffusely within the cytosol of the microvillar lobe, after exposure to PMA the fluorescence forms a much more pronounced ring around the edges and is nearly absent from the central portion (calibration bar: 5 µm). A quantitative index of immunofluorescence distribution was obtained by determining its intensity along a line, as indicated; the resulting profiles are plotted on the right. B, the bar graph shows the ratio Fcyt/Fmem (i.e. peak vs. trough of the profile) averaged for separate groups of cells in the two conditions. Error bars indicate standard deviation The difference was statistically significant by a Student's t test (P < 0.01, n = 17 and 20, respectively).

We exploited the translocation of PKC as an independent criterion to assess whether PKC is activated by light. Dark-adapted photoreceptors plated onto coverslips were either directly fixed in paraformaldehyde, or briefly illuminated immediately before immersion in the fixative. The intensity of illumination (~4.6 10^-3 W cm^-2) and its duration (5 s) were chosen to be comparable to the parameters used in background adaptation experiments (see below). After fixation, cells were processed for immunocytochemistry. Figure 7A shows bright-field and fluorescence micrographs of dissociated rhabdomeric photoreceptors under the two conditions. After illumination (bottom), the PKC signal appeared considerably more concentrated near the rhabdomeric membrane. An analysis of fluorescence intensity profiles was carried out as detailed above, and the results are shown in the bar graph in Fig. 7B. The difference in the average ratio F_cyt/F_memb between cells kept in the dark and those exposed to light was statistically significant (P < 0.01, Student's t test; n = 30 and 28, respectively). These observations indicate that stimulation with adapting lights activates PKC. Extending the post-illumination interval to 30 s before fixing the cells produced no further enhancement of PKC localization near the membrane (n = 11), suggesting that most of the spatial re-distribution takes place rapidly after light stimulation.

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Figure 7. Light stimulation induces translocation of PKC A, microvillar photoreceptors were dark adapted and then either fixed directly (top), or briefly illuminated (5 s, 10.4 10-3 W cm-2) prior to fixation (bottom). Illumination caused PKC to be primarily localized near the membrane of the rhabdomeric lobe. Calibration bar: 10 µm. B, the bar graph shows the average ratio Fcyt/Fmem for cells maintained in the dark (left, n = 30) vs. stimulated with a 5 s light and fixed either immediately (middle, n = 28) or after an additional interval in the dark (right, n = 11). Error bars indicate standard deviation; the difference between dark-adapted and light-stimulated cells was significant at the 0.01 level (Student's t test), whereas no significant difference was found between the two light-stimulation conditions.

Effects of PKC inhibitors on basal sensitivity

Having demonstrated that photostimulation activates a PKC in the light-sensitive microvillar lobe, and that chemical activation of PKC depresses responsiveness, we examined the physiological effects of pharmacological antagonists of this enzyme. Bisindolylmaleimides are a family of compounds that specifically inhibit PKC; among them, the derivative Ro-32-0432 (also known as bisindolylmaleimide XI) exhibits a great selectivity for PKC over other kinases (> 3 orders of magnitude), and a 3- to 10-fold preference for the isoform over other PKC isozymes (Birchall et al. 1994); furthermore, it presents the advantage of being cell-permeant. We first examined the effects on basal light sensitivity, by recording the current evoked by brief flashes of varying intensity separated by a prolonged dark adaptation interval. Collecting two full intensity series (before and during application of the antagonist) proved too taxing for most photoreceptors, as the light response often deteriorated before completion of the test; therefore, comparisons were made across groups, each cell only being tested in one condition. Figure 8A shows a semi-log plot of the average intensity response measured in ASW and during local application of 1 µM Ro-32-0432. The PKC inhibitor caused a ~0.6 log shift in the curve to the left. The mean half-saturating light intensity, determined by interpolation from individual intensity-response curves, is shown in the bar graph in Fig. 8B; a Student's t test confirmed that the difference was statistically significant (P < 0.05). Another PKC inhibitor, chelerythrine (5-20 µM), also sensitized the photocurrent elicited by flashes of fixed intensity (not shown). The modest, but reproducible enhancement in light sensitivity induced by Ro-32-0432 is compatible with the notion that some basal level of PKC activity is present in the dark, and is quenched by the antagonists.

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Figure 8. Application of a PKC inhibitor increases basal sensitivity of the light response A, intensity-response curves, averaged for two groups of cells, one tested in control conditions, and the other superfused for > 10 min with 1 µM Ro-32-0432 (n = 4-11 per point; notice that, for clarity, error bars indicate S.E.M.). Unattenuated light intensity 9.3 10-3 W cm-2. B, bar graph illustrating the average light intensity that elicited a half-maximal photocurrent in control cells and in Ro-32-0432-treated cells. The difference was significant at the 0.05 level (error bars indicate standard deviation).

Effects of PKC inhibitors on light adaptation

A more pertinent question regarding the possible involvement of PKC in light adaptation is whether pharmacological inhibitors can antagonize the desensitizing effects of conditioning and background lights. Because of the observation that light stimulates PKC, as determined by its translocation, one would indeed expect an enhanced susceptibility to PKC antagonists in the light-adapted state. We first tested the effects of Ro-32-0432 using a background adaptation protocol, in which a 100 ms test stimulus of fixed intensity was presented either alone, or superimposed upon steady illumination (BKG, see Fig. 9) that was turned on 5 s before the test flash. Intensities were chosen so that the test response was sub-saturating, and its amplitude was decreased ~30-70 % by the background light. Figure 9A shows photocurrents measured in dark-adapted conditions (D.A.), or superimposed on the sustained light (L.A.). The procedure was conducted in control solution (top panel) and repeated, in the same cell, after ~15 min of continuous puffer application of the PKC inhibitor (1 µM; bottom). Cells that displayed significant deterioration (> 20 %) in the amplitude of the light response to a control flash at the beginning vs. the end of the whole experiment were discarded. As noted before, the sustained illumination strongly adapted the photoreceptor, causing the light-evoked current to decline virtually to the baseline level. When the test flash was superimposed on the steady light, an incremental response was elicited but, as expected, its amplitude was much reduced, compared to dark-adapted conditions. In the presence of the drug the response to the adapting light also declined, but the degree of background-induced attenuation of the test-flash response was significantly reduced. Therefore, although during treatment with Ro-32-0432 the cell was still able to desensitize, its susceptibility was decreased. Notice also the changes in the relative peak amplitudes of the responses elicited by the adapting light and by the superimposed flash: in control conditions the transient current at the onset of the background (presented after extensive dark adaptation) was larger than the subsequent incremental flash response, whereas during the pharmacological treatment the size of the latter grew to exceed the background response; this argues against an indiscriminate enhancement of sensitivity, unrelated to the state of adaptation. Moreover, because the responses to the background and the incremental test flash were both significantly smaller in amplitude than the dark-adapted test photocurrent, the effect cannot be accounted for by response saturation. The bar graph in Fig. 9B shows the average ratio of light-adapted vs. dark-adapted test response amplitude for 11 cells subjected to the same protocol; the difference in the two conditions was statistically significant (P < 0.01; Student's t test for paired samples).

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Figure 9. Inhibitors of PKC reduce adaptation by background illumination A, responses to a 100 ms flash of fixed intensity (-3.5 log) were measured in dark-adapted conditions (D.A.), or in the presence of a sustained light (L.A.; -4.3 log). After an initial measurement in control solution (upper panel), the puffer pipette was lowered and the cell was continuously superfused with 1 µM Ro-32-0432 for ~15 min, before repeating the test (lower panel). In the presence of the PKC inhibitor the attenuation of the photoresponse by the background light was reduced. Vh = -50 mV. B, bar graph depicting the mean ratio of light-adapted vs. dark-adapted amplitude of the response to the test flash in the two conditions. Error bars indicate standard deviation. The difference was statistically significant (t test for paired samples, P < 0.01; n = 11).

Light adaptation was also examined using a conditioning-light protocol, in which a stimulus was delivered either after a long dark adaptation period (7 min), or shortly following another, identical stimulus (30-60 s). The temporal separation between pairs of stimuli was designed to produce a moderate degree of adaptation (see above). Cells that exhibited a rundown of > 20 % during the course of the whole experimental procedure were discarded from the analysis. Either brief flashes (100 ms; 10 cells) or 1 s lights (four cells) were used; because no differences were observed in the outcome of the two protocols, the data were pooled in the analysis. After a measurement in control conditions, the puffer pipette was lowered to superfuse the same cell with 1 µM Ro-32-0432 for 15 min, and the procedure was repeated. Figure 10A shows a set of traces from a representative photoreceptor. In the presence of the PKC inhibitor (bottom traces), the photocurrent evoked by the second stimulus of the pair was considerably less attenuated than in control conditions (top traces). The bar graph in Fig. 10B shows the ratio of the light-adapted/dark-adapted response amplitude in control conditions and during application of Ro-32-0432, averaged for the entire group of cells (n = 14). A Student's t test for paired samples indicated that the difference was statistically significant (P < 0.05). In order to rule out that these results may be accounted for by a gradual loss in the cell's susceptibility to light adaptation (e.g. stemming from the washout of some intracellular factor during prolonged intracellular perfusion), control tests were done in which the conditioning-adaptation protocol was applied twice, 30 min apart; no loss in the ability to adapt was observed (n = 9).

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Figure 10. PKC inhibition decreases the desensitization produced by a conditioning light A, light stimuli of constant intensity (100 ms, -2.6 log) were either presented after a 7 min dark-adaptation period (left), or 30 s following another stimulus (right), when the cell sensitivity had not yet fully recovered. The procedure was repeated in the same photoreceptor cell in the presence of 1 mM Ro-32-0432, applied by puffer pipette. Response attenuation induced by the preceding conditioning flash was smaller after treatment with the PKC inhibitor. B, the bar graph shows the average degree of light adaptation (ratio of peak amplitude of photoresponses elicited 30 s vs. 7 min after the previous flash), in control conditions and during subsequent treatment with Ro-32-0432. Error bars indicate standard deviation; the difference was statistically significant at the 0.01 level (Student's t test for paired samples, n = 14). C, reduction of adaptation by conditioning lights, induced by intracellular perfusion with 50 mM of an inhibitory peptide, PKC fragment 19-36. The comparison was made across separate groups of cells, and the difference was statistically significant (P < 0.05; n = 6 and 8 for the experimental and the control group, respectively). Holding potential -50 mV.

Finally, a similar conditioning adaptation protocol was used to test the effects of a different PKC inhibitor, a synthetic peptide formed by residues 19-36 of PKC. This fragment comprises the pseudo-substrate domain that binds to the active site of the catalytic domain when the enzyme is in the resting state (Hannun et al. 1985). Because the peptide was applied intracellularly by dialysis via the patch pipette, the comparison was done across cells. As shown in the bar graph in Fig. 10C, photoreceptors treated with 50 µM PKC(19-36) (n = 6) exhibited a significantly reduced adaptation by the conditioning light (t test, P < 0.05) compared to control cells (n = 8).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Elevation of cytosolic Ca²⁺ has long been postulated to be a key component of the light adaptation process (Lisman & Brown, 1972, 1975). Although the mode of action of Ca²⁺ is not fully understood, several leads point to PKC as one of its possible downstream effectors. We showed that in molluscan microvillar photoreceptors stimulation with DAG surrogates markedly reduces light responsiveness, the effect being specific to those visual cells that utilize a PLC-based transduction cascade. The existence of an intimate relation between PKC and the light-signalling pathway is supported by its association with InaD and the phototransduction complex (Tsunoda et al. 1997; Adamski et al. 1998; Xu et al. 1998). We established the presence of a PKC-like isozyme in rhabdomeric photoreceptors, primarily confined to the light-transducing lobe. PKC belongs to the sub-set of 'conventional' or 'classical' PKCs, (cPKCs; reviewed by Newton, 1997), which possess a C1 domain that binds DAG, and a C2 domain that binds Ca²⁺, and both agents can function as activators. Because in rhabdomeric photoreceptors light triggers the production of DAG and elevates [Ca²⁺]_i, a possible pathway for PKC regulation is readily envisioned. Also, the Ca²⁺-binding C2 domain in PKC could account for the ability of an imposed Ca²⁺ elevation to initiate photoresponse desensitization in the absence of illumination (and thus without a light-triggered increase in cellular DAG).

In the dark, PKC immunofluorescence is distributed throughout the rhabdomeric lobe, with some accumulation near the membrane. Upon illumination, the spatial segregation of PKC becomes significantly more pronounced; this translocation from the cytosol to the membrane provides independent confirmation of activation by light. The effect appears to be rapid, because the cells were quickly dipped into the fixative immediately after the termination of a 5 s light. Translocation of PKC on the time scale of seconds has been previously reported in other systems (e.g. Cesare et al. 1999). Of course, the actual fixation time cannot be determined with any accuracy, but ambiguities about the time course of translocation have been entirely circumvented in studies that used heterologous expression of PKC-Green Fluorescent Protein (GFP) fusion constructs. This strategy permits real-time visualization of PKC in live cells (Sakai et al. 1997), and revealed that PLC stimulation can induce a rapid, reversible redistribution of PKC-GFP, capable of tracking Ca²⁺ spikes with a lag as brief as ~1 s (Oancea & Meyer, 1998). Therefore, the rapidity of PKC translocation appears to be compatible with that of light adaptation, which sets in with a time constant of hundreds of milliseconds. We also observed an enhancement of PKC localization in the rhabdomeric membrane after stimulation with 1 µM PMA, a result that dovetails with the desensitizing effects of the same treatment on the light response.

Pharmacological antagonists of PKC caused a modest, but reproducible enhancement of sensitivity in the dark-adapted state, as also reported in Limulus (Dabdoub & Payne, 1999). The effect was obtained with Ro-32-0432, an agent that preferentially targets -type PKCs (Birchall et al. 1994), and may reflect quenching of the basal activity of PKC. The existence of a background level of PKC activity is compatible with the observation that a fraction of the enzyme localizes near the membrane even in the dark. The most interesting effect of PKC inhibitors, including a peptide comprising the autoinhibitory domain of the enzyme, was their ability to antagonize the desensitization induced by either background illumination or conditioning flashes. The sensitizing action was preferentially strong on light-stimulated, as opposed to dark-adapted cells, as reflected in the changes in the light-adapted:dark-adapted response amplitude ratio. Such an outcome is consistent with the susceptibility of cPKCs to be stimulated by Ca²⁺ and DAG, the two prime messengers mobilized by illumination. Pharmacological rescue of sensitivity during light-adapting conditions required adapting lights that produced a moderate depression of the test response. By contrast, the use of excessively strong adapting stimuli (i.e. nearly abolishing the test response) often failed to reveal a significant recovery of sensitivity by PKC inhibitors, an outcome that could reflect saturation of the adaptation process. This may be one factor underlying the reported failure of another bisindolylmaleimide derivative to recover the flash response after strong desensitization caused by a conditioning light in Limulus ventral photoreceptors (Dabdoub & Payne, 1999).

Our results supporting a role for cPKC in the desensitization of the visual response in Lima are consistent with the proposition that in the Drosophila PKC-deficient mutant, InaC, the shift in sensitivity by background illumination is attenuated (Hardie et al. 1993). Such an apparent adaptation deficit has not been easy to isolate unambiguously, because of the complexity of the InaC phenotype, which, paradoxically, also includes an enhanced tendency for the photoresponse to decay during sustained light (Hardie et al. 1993), and a slower recovery after conditioning flashes (Smith et al. 1991). The downstream targets of PKC in light adaptation remain to be elucidated. PKC phosphorylates InaD and trp in Drosophila (Huber et al. 1996; Liu et al. 2000), but additional potential candidates exist along the light transduction pathway: electrophysiological measurements in Limulus suggest that DAG analogues antagonize an early step of the cascade (Dabdoub & Payne, 1999). In other cell types PKC can phosphorylate G_q (Aragay & Quick, 1999), PLC- (Ali et al. 1997; Strassheim et al. 1998) and InsP₃Rs (Ferris et al.1991). A role for PKC in sensory adaptation has also been documented in olfactory neurons (Boekhoff & Breer, 1992) and has been proposed for taste receptors (Ozaki & Amakawa, 1992), suggesting that such a mechanism may be of wide generality in sensory physiology.

The roles of PKC are ubiquitous, and a host of other processes are likely to be PKC-dependent in photoreceptors. For example, in arthropod eyes PKC activators also trigger the shedding of the photosensitive membrane (Blest et al. 1992; Jinks et al. 1996). This circadian event is presumably mediated by activation of a PKC II, as suggested by immunostaining (Jinks et al. 1996). Although a transient loss of rhabdomeral membrane could be accompanied by a reduction in sensitivity, this process is slow: in Limulus lateral eyes, structural changes of the rhabdomere occur ~30 min after stimulation with high concentrations (millimolar) of phorbol esters (Jinks et al. 1996). By contrast, we observed translocation of PKC and desensitization of the photocurrent by DAG surrogates over a time scale of seconds, which is much too rapid for membrane turnover. Naturally, our proposal of PKC involvement in the rapid modulation of the photoresponse with changing ambient illumination by no means disputes any additional role of other PKCs in longer-term cellular regulation, such as the turnover of the photosensitive membrane.

PKC cannot be the sole effector controlling light adaptation, since alternative Ca²⁺-dependent pathways can regulate the photocurrent. In the first place, high Ca²⁺ concentrations are also known to antagonize InsP₃ receptor/channels in the endoplasmic reticulum, (Bezprozvanny et al. 1991). Activation of InsP₃ receptors contributes to the light-evoked increase in [Ca²⁺]_i, at least in some microvillar photoreceptors, and elevated Ca²⁺ downregulates the light-evoked Ca²⁺ release (Payne et al. 1990; Levy & Payne, 1993). This negative feedback process could result in a decreased transduction gain, owing to the fact that Ca²⁺, in addition to its slow desensitizing effects, also exerts a rapid synergistic action on the excitatory process (Bolsover & Brown, 1985; Payne et al. 1986; Payne & Fein, 1986; Werner et al. 1992; Shin et al. 1993). Of course, such a mechanism may be of marginal relevance to species that seem to lack a significant light-elicited Ca²⁺ release from internal stores (Brown & Blinks, 1974; Ranganathan et al. 1994), or in which the InsP₃R appears not to be critical for light responsiveness (Acharya et al. 1997; Raghu et al. 2000). In the second place, in analogy with vertebrate rods, direct blockade of light-sensitive channels by Ca²⁺ and/or changes in their sensitivity to the internal transmitter are also likely: in fact, the photocurrent can be downregulated by Ca²⁺ influx through the light-activated conductance (Hardie, 1991), or photorelease of caged Ca²⁺ (Hardie, 1995b). Finally, desensitization is not the sole consequence of light adaptation: background adaptation is typically accompanied by photoresponse acceleration. The functional consequence of such a phenomenon is that when the impinging photons are few, a long integration time improves signal-to-noise ratio (at the expense of speed) by enhancing summation of quantal events, whereas at high levels of ambient light the photon flux is not a limiting factor, and it is advantageous to optimize photoresponse rapidity. Remarkably, we have failed to observe a speeding up of the flash response concomitant with the desensitization induced by PKC activators (Gomez & Nasi, 1998). A similar outcome was also reported by Dabdoub & Payne (1999), leading these authors to question a physiological role for PKC activation in light adaptation (even though its powerful desensitizing effects were corroborated). Because stimulation of conventional PKCs in the microvillar lobe seems an obligatory consequence of the light-triggered Ca²⁺ elevation and DAG production, we propose instead an alternative explanation: downstream effectors of Ca²⁺ may diverge, and while PKC may be involved in the modulation of sensitivity, the changes in response kinetics are likely to be under the control of separate, as yet unidentified, signalling pathways. Recent observations in fact provide clear indication that, at least in some systems, the two processes can be dissociated (Nasi & Gomez, 1999).

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Acknowledgements

We thank Dr Wayne Sossin for providing the anti-Aplysia PKC antiserum. Supported by grant RO1-EY07559 from the National Institutes of Health, USA.

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