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The Journal of Neuroscience, December 1, 1999, 19(23):10262-10269
Protein Kinase C Activators Inhibit the Visual Cascade in
Limulus Ventral Photoreceptors at an Early Stage
Alain
Dabdoub and
Richard
Payne
Department of Biology, University of Maryland, College Park,
Maryland 20742
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ABSTRACT |
The phosphoinositide cascade mediates visual transduction in
invertebrate photoreceptors. Phospholipase C (PLC) catalyzes the
hydrolysis of phosphatidylinositol bisphosphate, producing inositol
trisphosphate (InsP3) and diacylglycerol (DAG).
Protein kinase C (PKC) is a major target of DAG in many cell types. We have used PKC activators to investigate the function of the kinase in
the phototransduction cascade in Limulus polyphemus
ventral photoreceptors. Extracellular application of ( )-indolactam V (0.03-30 µM) or phorbol-12,13-dibutyrate (10 µM) reversibly reduced the sensitivity of the electrical
response of the photoreceptors to light by up to 1000-fold. The inert
stereoisomer (+)-indolactam V and 4 -phorbol had no effect. The
effect of ()-indolactam V was antagonized by the PKC inhibitors
bisindolylmaleimide I and Gö 6976. Coapplication of
bisindolylmaleimide V, used as a negative control compound for PKC
inhibition, did not reduce the effectiveness of ()-indolactam V. These findings are consistent with ()-indolactam V activating PKC and
desensitizing the light response. Furthermore, our pharmacological
results indicate that PKC activation does not appear to play a role in
light adaptation. We localized the position of the target of PKC in the
visual cascade. We chemically excited the cascade at various stages to
determine the kinase's target. PKC activation by ()-indolactam V
decreased the light-induced elevation of intracellular calcium but had
no effect on the photoreceptor's excitatory response to intracellular
injection of InsP3. However, the PKC activator greatly
reduced the excitation caused by GTP- -S injection. We propose that
PKC inhibits the visual transduction cascade at the G-protein and/or
PLC stage.
Key words:
Limulus polyphemus; photoreceptor; PKC; phototransduction; phosphoinositide pathway; visual inhibition; desensitization; adaptation
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INTRODUCTION |
In invertebrate microvillar
photoreceptors, absorption of light by rhodopsin activates
phospholipase C (PLC) via a GTP-binding protein (Bloomquist et
al., 1988 ; Ranganathan et al., 1995). PLC catalyzes the hydrolysis of
phosphatidylinositol bisphosphate producing inositol trisphosphate
(InsP3) and diacylglycerol (DAG) (Berridge,
1993). The role of the DAG branch of the cascade is not clear. In many
cell types DAG is an activator of the enzyme protein kinase C (PKC)
(Nishizuka, 1988), which in turn phosphorylates a variety of
targets. PKCs make up a family of serine/threonine kinases that are
involved in the regulation of many cellular processes.
Light initiates three general processes in invertebrate microvillar
photoreceptors: the activation of cation channels in the microvillar
membrane (excitation), desensitization of the visual cascade
(adaptation), and long-term changes in the structure of the microvillar
membrane, such as the shedding and renewal of rhabdom at dawn. PKC
has been proposed to play a role in all of these processes.
Application of PKC activators to photoreceptors of the clam
Lima evokes an inward current that shares some of the
characteristics of the light-induced current (Gomez and Nasi, 1998).
Studies of the Drosophila mutant
inactivation-no-afterpotential C (inaC), which lacks an
eye-specific PKC (Smith et al., 1991), have indicated a role for PKC in
adaptation and response deactivation (Smith et al., 1991; Hardie et
al., 1993). In the horseshoe crab Limulus polyphemus, PKC
activators induce rhabdom shedding when injected into the compound
lateral eye before dawn (Jinks et al., 1996). PKC activators have also
been reported to reduce potassium channel activity and promote changes
in photoreceptor morphology in the marine mollusc
Hermissenda (Farley and Auerbach, 1986; Lederhendler et al.,
1990; Etcheberrigaray et al., 1992).
Recent biochemical experiments have demonstrated putative PKC activity
in Limulus photoreceptors. The addition of calcium and
phospholipid to homogenates of Limulus ventral and lateral eyes resulted in the phosphorylation of several proteins, and this
phosphorylation was inhibited by the PKC inhibitor peptide PKC 19-36
(Calman et al., 1996). The giant ventral nerve photoreceptors of
Limulus have several advantages for a pharmacological study of the action of PKC. The physiological and anatomical consequences of
illumination have been well defined. In Limulus ventral
photoreceptors, light-induced production of InsP3
causes the release of calcium from intracellular stores (Brown and
Rubin, 1984; Payne et al., 1986b), leading to the opening of
nonspecific cation channels in the plasma membrane and a resulting
depolarization of the photoreceptor's membrane potential. Calcium
release subsequently reduces the photoreceptors' sensitivity,
initiating light adaptation (Brown and Lisman, 1975). Prolonged bright
illumination also leads to profound, reversible changes in rhabdom
structure (Herman, 1991). The stability of electrophysiological
recordings from isolated ventral photoreceptors permits investigation
of the reversibility of the effects of PKC activators, whereas the
response to pulsed intracellular injection of intermediates, such as
InsP3, allows the identification of the level of
the visual cascade at which phosphorylation by PKC might act.
Parts of this paper have been published previously (Dabdoub and
Payne, 1998).
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MATERIALS AND METHODS |
Preparation of the ventral nerve photoreceptors was performed as
described by Millecchia and Mauro (1969a,b). Nerves were placed in
artificial seawater (ASW) that contained (in mM): 435 NaCl,
10 KCl, 20 MgCl2, 25 MgSO4,
10 CaCl2, and 10 HEPES at pH 7.3. Phorbol-12,13-dibutyrate (PDBu), 4-phorbol, bisindolylmaleimide I
and V, and Gö 6976 were obtained from Calbiochem (San
Diego, CA). ()-Indolactam V was obtained from both Calbiochem and
Sigma (St. Louis, MO), and (+)-indolactam V was obtained from Sigma. All these substances were dissolved in DMSO as a 5-10 mM
stock solution and kept at 20°C. Test substances were applied
extracellularly by superfusion (1 ml/min; 5-10 times bath vol)
followed by a 10 min wait before continuing with the experiment.
White light from a 100 W quartz-halogen source (model 6333; Oriel
Corporation, Straford, CT) illuminated the photoreceptors. The light
passed through a heat filter (Schott KG3; Ealing Optics, South Natick,
MA), neutral density (ND) filters, and a shutter before being focused
onto the specimen plane. The intensity of the light at the specimen,
with no intervening ND filters, was 80 mW/cm2. Light intensities are quoted in
this paper as log10 units of attenuation relative
to this intensity. To view the preparation with an infrared-sensitive
video camera, we also continuously illuminated cells by an infrared
beam, created by passing a second beam of light from the
quartz-halogen lamp through an infrared filter (Schott RG1000) before
focusing it onto the specimen. Attenuation of the light by a
log10 8.5 ND filter resulted in a rate of one single photon event per second.
For intracellular recording, the micropipette contained 3 M
KCl. For voltage-clamp experiments, cells were impaled with a second
microelectrode that also contained 3 M KCl (resistance < 10 M ) and were voltage clamped at the dark resting membrane potential using an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). The voltage-clamped current or the membrane potential was
filtered at 300 Hz and sampled at 1 kHz using a Digidata 1200 (Axon
Instruments) analog-to-digital board installed in a personal computer.
The pCLAMP (Axon Instruments) software programs Clampex and Fetchex
(version 6.0.3) were used for data acquisition.
All chemicals injected into the cell were dissolved in a carrier
solution that contained 100 mM potassium aspartate and 10 mM HEPES, at pH 7.0. Injections were monitored using the
infrared video camera (Corson and Fein, 1983a).
InsP3 was obtained from Research Biochemicals
(Natick, MA), dissolved into a 10 mM stock solution, and
kept at 20°C. Poorly hydrolyzable
L-Ins(1,3,4)P3 was the gift of Dr.
Barry Potter (University of Bath, Bath, United Kingdom). GTP--S was
obtained from Sigma, dissolved into a 1 mM stock solution,
and kept at 70°C. For excitation by InsP3, 10 or 100 µM InsP3 in carrier solution
was pulse pressure injected into the light-sensitive rhabdomeral lobe
of the photoreceptor as described by Fein et al. (1984). For activation
of G-proteins, 100 µM GTP--S in carrier solution was
pressure injected into the cell. After multiple injections, nucleotide
exchange was activated by light flashes after which the photoreceptor
was dark adapted (Bolsover and Brown, 1982).
Experiments using confocal imaging and a calcium fluorescent indicator
were performed as described previously (Ukhanov and Payne, 1995).
Briefly, cells were injected with 500 µM Oregon green-5N
(Molecular Probes, Eugene, OR), a fluorescent calcium indicator dye, in
carrier solution. The dissociation constant Kd of Oregon green-5N was determined to be
18 µM in a solution containing 400 mM KCl and 10 mM HEPES at
pH 7.0. The photoreceptors were viewed with a Zeiss LSM 410 laser-scanning confocal microscope equipped with a 488 nm argon laser
(Uniphase) focused through a Zeiss Neofluor 10×, 0.3 numerical
aperture objective lens using the stationary spot recording mode
of the microscope.
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RESULTS |
Activators of PKC inhibit the electrical response to light
()-Indolactam V is a potent and selective activator of PKC that
serves as a mimic of the endogenous activator DAG. ()-Indolactam V
activates PKC by binding to the same site on the enzyme as phorbol esters (Heikkilä and Åkerman, 1989). The specificity of
()-indolactam V can be verified by performing control experiments
using the stereoisomer (+)-indolactam V. Figure
1 illustrates the effects of addition of
both isomers. A ventral nerve photoreceptor was dark adapted and
voltage clamped to its resting membrane potential. A 1 sec light flash
(intensity, log10 3) excited an inward current, which peaked at an amplitude of 160 nA and then declined as
adaptation proceeded (Fig. 1A, top).
Superfusing the cell with the inactive stereoisomer (+)-indolactam V
(25 µM) had no effect on the photoresponse (Fig. 1A, middle). Superfusing the cell
with the same concentration of the active stereoisomer ()-indolactam
V reduced the peak response amplitude to 7 nA and increased its
latency (Fig. 1A, bottom). ()-Indolactam
V affected all photoreceptors tested in a similar manner. The reduction
of sensitivity induced by 25 µM ()-indolactam V was analyzed in another cell by plotting the relationship between the
peak response and light intensity. The effect of 25 µM ()-indolactam V could be described
approximately as a shift of the intensity-peak response curve to the
right by 2.5 log10 units coupled with a reduction of the saturated light-induced current by 0.8 log10 units (Fig. 1B,
n = 4). The effects of ()-indolactam V were partially reversible after a 2 hr wash.
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Figure 1.
()-Indolactam V reversibly and
stereospecifically inhibits the light response causing a shift in the
intensity-response curve. A, The photoreceptor's
response to a 1 sec light stimulus (log10 3) under
voltage-clamp conditions in ASW (top) is unaffected by
25 µM (+)-indolactam V, the inactive stereoisomer
(middle). The response to that same stimulus is strongly
reduced (7 nA) in the presence of 25 µM ()-indolactam
V (bottom). B, ()-Indolactam V at 25 µM causes a shift in the intensity-response curve to the
right by ~2.5 log10 units and a reduction
in the saturated light-induced current of ~0.8 log10
units. The effects of ()-indolactam V were partially reversible after
2 hr of washing in ASW. The light stimulus was 1 sec long, delivered at
1 min intervals.
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In current-clamp recordings of membrane potential, ()-indolactam V
decreased the photoresponse in a dose-dependent manner. Receptor
potentials elicited by a 20 msec light flash
(log10 5.4) decreased with increasing
()-indolactam V concentrations (Fig. 2A). Desensitization of
cells after application of ()-indolactam V was not accompanied by
significant (>4 mV) changes in resting membrane potential or a
reduction in electrical input resistance, measured with brief 0.3-3
nA current pulses. To check whether inhibition of the photoresponse is
a common characteristic of PKC activators, we used the phorbol ester
PDBu, a chemically distinct molecule that is also a specific PKC
activator. Extracellular application of 10 µM
PDBu inhibited the photoresponse in a manner similar to that of
()-indolactam V (Fig. 2B, n = 4).
The effects of PDBu persisted for hours and were partially reversible
after an 8 hr wash (data not shown). The inert 4-phorbol had no
effect on the photoresponse (n = 3), further confirming
the specificity of the effects of PKC activators.
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Figure 2.
PKC activators decrease the photoreceptor's
sensitivity to light. A, The receptor potential, under
current-clamp recording, decreases with increasing ()-indolactam V
concentrations. B, PDBu also decreases the receptor
potential, whereas the inactive 4-phorbol has no effect. The
horizontal bar below the
trace represents the duration of the light flash (20 msec; log10 5.4). Compounds were dissolved in DMSO and
applied extracellularly to the bath. The percent DMSO was 0.18% for
the different concentrations of ()-indolactam V, 0.2% for PDBu, and
0.1% for 4-phorbol.
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We examined whether the inhibition caused by DAG surrogates was indeed
the result of PKC activation. We pretreated the photoreceptors with PKC
inhibitors and tested the cell's response to ()-indolactam V. We
used bisindolylmaleimide I, a selective PKC inhibitor that acts as a
competitive inhibitor to the ATP-binding site of PKC (Toullec et al.,
1991). As a control, we used bisindolylmaleimide V (Davis et al.,
1992). We also used Gö 6976, an indolocarbazole, another highly
potent and selective inhibitor of PKC (Martiny-Baron et al., 1993).
For these experiments, we decided to monitor the sensitivity of the
cell under current clamp, rather than two-electrode voltage clamp.
Impaling the cell with a single micropipette minimized damage to the
cell, resulting in less variation in the dark-adapted sensitivity to
light and a greater chance that sensitivity would be stable for long
time periods. However, the use of current-clamp recording complicates
the analysis of the responses. For dark-adapted Limulus
ventral photoreceptors, the relationship between receptor potential and
flash intensity is highly nonlinear. Voltage-sensitive conductances are
active even during single photon signals of >10 mV in amplitude. Above
this depolarization, rectification and the approach to the reversal
potential compress the intensity-response curve (Stieve and Pflaum,
1978). This means that a small change in receptor potential amplitude
might reflect a large change in the sensitivity of the cell. Rather
than measure receptor potential amplitude in response to flashes of
fixed intensity, we therefore quantified the effects of the inhibitors
by monitoring the sensitivity of the cell as the inverse of the
intensity of a 20 msec flash required to elicit a 30 mV criterion
response. The effects of voltage-dependent conductances are then the
same for all the measurements, and changes in the sensitivity of the
criterion response should reflect changes in the sensitivity of the
underlying photocurrent, which displays a much larger linear dynamic
range. This seems to hold for our data. The 3.3 log10 unit decrease in sensitivity to a 30 mV
criterion response induced by 30 µM
()-indolactam V (Fig. 3B)
can be compared with the 2.5 log10
unit reduction in the voltage-clamp current after a 3
log10 unit flash induced by 25 µM ()-indolactam V (Fig.
1B).
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Figure 3.
The effectiveness of ()-indolactam V is
decreased by selective PKC inhibitors. A, The PKC
inhibitors bisindolylmaleimide (BIM)
I and Gö 6976 significantly reduced the
effectiveness of ()-indolactam V when either was coapplied.
BIM V, a negative control compound for PKC inhibition,
did not alter the effectiveness of ()-indolactam V. The
horizontal bar below the
bottom receptor potential traces
represents the duration of the light flash (20 msec); the intensity of
the flashes was the same before and after application of 1 µM ()-indolactam V. B, Changes in
sensitivity to a criterion response induced by ()-indolactam V ( )
were measured. A 30 mV criterion response to a 20 msec light flash was
chosen, and the change in light intensity required to evoke the
criterion response was measured (0.5% DMSO at all concentrations). At
a much higher concentration, the inactive stereoisomer (+)-indolactam V
( ) had no effect on the photoreceptor's sensitivity to light (1.8%
DMSO). C, The change in the photoreceptor's sensitivity
was measured for the same criterion response in the presence of
specific PKC inhibitors as well as different concentrations of
()-indolactam V. Sensitivity changes are shown relative to that
measured after application of the inhibitor but before application of
()-indolactam V and therefore do not reflect a small sensitization
induced by the inhibitor alone.
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Without addition of inhibitor, sensitivity decreased progressively as
()-indolactam V concentration increased from 0.03 to 30 µM (Fig. 3A, top, B;
n = 4-6 cells for each concentration). The inactive
stereoisomer (+)-indolactam V had no effect (Fig. 3B,
n = 3). The dependence of sensitivity to light on
()-indolactam V concentration was then reexamined in the presence of
the two PKC inhibitors (Fig. 3A,C). The PKC inhibitors alone
induced a small increase in the photoreceptors' sensitivity (0.5 ± 0.4 log10 units; n = 9, pooled
data using either inhibitor). The inhibitors greatly reduced the
effectiveness of ()-indolactam V (Fig. 3A,C; n = 4-5 for each group). In comparison with cells that
were not bathed in inhibitors (Fig. 3B), the relative
sensitivity was increased by 1.3 log10 units in
the presence of 1 µM ()-indolactam V and by
2.2 log10 units in the presence of 10 µM ()-indolactam V. We obtained similar
results using chelerythrine and calphostin C, a PKC inhibitor that
interacts with the kinase's regulatory domain by competing at the
binding site of DAG and phorbol esters (data not shown).
Bisindolylmaleimide V had no significant effect on the desensitization
caused by ()-indolactam V (compare Fig. 3B,C). Taken
together, these results are consistent with ()-indolactam V
activating PKC and desensitizing the photoresponse.
PKC inhibitors do not antagonize light adaptation
We investigated the effects of the PKC inhibitor Gö 6976 on
the kinetics of the electrical response to sustained bright light and
on light adaptation. Light adaptation was investigated by delivering
paired 20 msec light flashes to voltage-clamped, dark-adapted photoreceptors. The response to the second flash in the pair was greatly reduced by light adaptation (ASW, first flash, 202 ± 5 nA;
second flash, 11.3 ± 0.6 nA; n = 3; Fig.
4A). Superfusion with
ASW containing 25 µM Gö 6976 resulted in
a relatively small but significant (t test,
p < 0.05) increase in the peak response to the first
flash, consistent with the increase in the sensitivity of dark-adapted
photoreceptors described above. However, the PKC inhibitor had no
significant effect (t test, p > 0.05) on
the amplitude of the light-adapted response to the second flash (25 µM Gö 6976, first flash, 247 ± 19 nA;
second flash, 8.7 ± 1.9 nA; n = 3; Fig.
4B). In other experiments, bisindolylmaleimide I had
effects on dark-adapted sensitivity similar to those shown in Figure 4,
whereas bisindolylmaleimide V had no significant effect (data not
shown).
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Figure 4.
PKC inhibitors sensitize the photoreceptor and do
not appear to affect light adaptation. A, Photocurrents
elicited by two 20 msec light flashes (log10 3.5; 1 sec
apart) under voltage-clamp conditions in ASW are shown.
B, The response to the first flash is increased in the
presence of 25 µM Gö 6976, whereas the second flash
is unchanged. C, Receptor potential elicited by a
prolonged intense light (log10 1), after overnight
incubation with 25 µM Gö 6976.
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We were concerned that in the experiment of Figure 4, A and
B, the slightly larger amplitude of the first, dark-adapted
response in the presence of the PKC inhibitor might also result in a
larger adapting effect, thus masking any relief of adaptation induced by the PKC inhibitor. Therefore in experiments on three cells, we
compensated for the increase in light-induced current after application
of 25 µM Gö 6976 by slightly reducing the
intensity of the light to maintain a 50 nA response to a test flash.
We also correspondingly reduced the intensity of a bright, adapting flash. We found that the adapting flash reduced the response to the
test flashes from 50 to 5 ± 0.25 nA in ASW and to 7 ± 3.5 nA in
the presence of the PKC inhibitor. Thus this modified protocol also
failed to demonstrate any significant relief of light adaptation by
Gö 6976. We also used adaptation protocols in which the
sensitivity of the photocurrent was examined in the presence of steady
background illumination. For background intensities of
log10 3 and log10 2,
which desensitized voltage-clamped photoreceptors by 2-3
log10 units, application of 25 µM Gö 6976 had no significant effect on
the peak amplitude of the light-adapted responses to test flashes (data
not shown).
The rapid transition during sustained bright illumination from the
initial peak transient phase of the response to the lower steady-state
phase is another consequence of light adaptation. In the
Drosophila inaC mutant the latter phase is missing; instead the response to sustained bright light decays slowly to baseline (Hardie et al., 1993). Ventral photoreceptors that were incubated overnight in 25 µM Gö 6976 displayed
receptor potentials in response to prolonged bright light
(log10 1) that still showed a rapid transition
from a transient phase to a sustained steady-state phase (Fig.
4C, n = 4). These responses are
indistinguishable from control responses, as described by others
(Millecchia and Mauro, 1969a).
()-Indolactam V inhibits the phototransduction cascade at the
G-protein and/or PLC stage
Because phosphorylation of ion channels by protein kinase C is a
well known mechanism for regulating channel activity, we examined
whether the PKC acted only at a late stage in the cascade, after
InsP3-induced calcium release. In this case, the
activators should not influence the amount of calcium released as a
result of light stimulation. To measure light-induced calcium release, we pressure injected ventral photoreceptor cells with the
calcium-sensitive fluorescent dye Oregon green-5N. Confocal spot
measurements of the fluorescence of Oregon green-5N were made in the
light-sensitive rhabdomeral lobe. The delayed increase in the
fluorescence of calcium-sensitive dyes observed during the first few
hundred milliseconds of confocal laser illumination has been shown
previously to indicate light-induced release of intracellular calcium
ions from intracellular stores (Ukhanov and Payne, 1995; Ukhanov et
al., 1995). Figure 5 shows the
simultaneous recording of the membrane potential (bottom) and Oregon green-5N signal (top). Measurements were first
taken before superfusion with ()-indolactam V (Fig. 5A).
Both the electrical response and the calcium signal decreased in
amplitude in the presence of 25 µM
()-indolactam V (Fig. 5B, n = 4). The
effects were partially reversible after a 2 hr wash (Fig.
5C). We conclude that ()-indolactam V inhibits the visual
cascade at a stage that is upstream from calcium release.
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Figure 5.
()-Indolactam V reduces light-induced calcium
release. Simultaneous recordings were made of membrane potential
(bottom) and the fluorescence of the calcium-indicator
dye Oregon green-5N (top). The cell was pressure
injected with the calcium indicator, and confocal spot measurements of
Oregon green-5N fluorescence were made in the light-sensitive
rhabdomeral lobe of the ventral photoreceptor. Membrane potential was
sampled every 5 msec, and fluorescence was sampled every 4.5 msec.
A, Photoreceptor bathed in ASW. B, After
treatment with ()-indolactam V. C, After a 2 hr wash
with ASW. arb., Arbitrary.
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We next determined whether PKC activation blocked
InsP3-induced calcium release. If this was the
case, then PKC activation should strongly inhibit the excitation caused
by intracellular injection of InsP3, just as it
inhibits the light response. Excitation by InsP3
injection was insensitive to 25 µM ()-indolactam V
(Fig. 6, n = 5). In
addition, 25 µM ()-indolactam V also had no
effect on repetitive bursts of depolarization caused by injecting 100 µM of the poorly hydrolyzable
InsP3 analog
L-Ins(1,3,4)P3 (data not
shown) (Riley et al., 1994). These results indicate that the primary
target of PKC is upstream from InsP3-induced
calcium release.
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Figure 6.
The desensitization of the photoreceptor by
()-indolactam V is upstream from the InsP3 receptor in
the cascade. Membrane potential was recorded during the delivery of an
intracellular pressure injection (100 msec) of 100 µM
InsP3 followed by a 20 msec light flash (log10
5). A, Photoreceptor bathed in ASW. B,
After treatment with 25 µM ()-indolactam V.
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Finally, we examined the effect of PKC activation on excitation caused
by the G-protein activator GTP--S (Pfeuffer and Helmreich, 1975).
Injecting the photoreceptor with 100 µM GTP--S
followed by exposure to light increases the frequency of discrete
events in the dark (Fig. 7A,C)
(Corson and Fein, 1983b). These events are smaller on average than
spontaneous or light-induced events (Fig. 7A,B), probably
because of the reduced amplification associated with the activation of
the G-protein rather than rhodopsin (Kirkwood and Lisman, 1994). The
induction of these events by GTP--S injection is irreversible, and
for large injections, the events fuse to form a noisy sustained
depolarization (Corson and Fein, 1983b). The amplitude of
GTP--S-generated events was reduced by superfusion with the PKC
activator ()-indolactam V (Fig. 7D, n = 4), reducing the level of noise in darkness. After a 2 hr wash with
ASW, the noise recorded in darkness increased, suggesting recovery of
the amplitude or frequency of GTP--S-generated events, but
individual events could not be distinguished (Fig. 7E). The
variance of this noise was transiently reduced from 0.16 to 0.06 mV2 after 20 msec flashes of light
(log10 4.4) consistent with the adaptation of
GTP--S-induced events (Fein and Corson, 1981). We therefore conclude
that desensitization by ()-indolactam V is downstream from
rhodopsin.
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Figure 7.
Desensitization by ()-indolactam V is downstream
from rhodopsin. A, Recording of membrane potential shows
spontaneous depolarizing discrete events recorded in darkness.
B, Light-induced discrete events (log10
8.4) are shown. C, The cell was injected with
GTP--S, and nucleotide exchange was activated by a series of light
flashes, after which the cell was dark adapted. This resulted in a
sustained increase in the frequency of small discrete events (*).
D, Extracellular application of ()-indolactam V
reduced the amplitude of the GTP--S-generated events.
E, After 2 hr of wash, the noise level of the
trace increased, consistent with the recovery of
GTP--S-induced events.
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DISCUSSION |
PKC modulates photoreceptor sensitivity to light
We have demonstrated that the light response of Limulus
ventral nerve photoreceptor cells can be reversibly desensitized by several orders of magnitude by DAG surrogates as was reported in
moluscan photoreceptors (Gomez and Nasi, 1998). We found this by using
two distinct and potent activators of PKC, ()-indolactam V, an indole
alkaloid, and PDBu, a phorbol ester. PDBu and ()-indolactam V did not
excite ventral photoreceptors or induce a membrane current, indicating
that PKC does not mediate excitation in these photoreceptors. This is
in contrast to Lima photoreceptors in which it was proposed recently that PKC mediates, at least in part, photoexcitation (Gomez
and Nasi, 1998). The lack of an effect of the inert stereoisomer (+)-indolactam V and 4-phorbol on photoreceptor sensitivity
establishes the specificity of the result. In addition, PKC inhibitors
antagonized the desensitization caused by ()-indolactam V, confirming
that the desensitization was indeed the result of PKC activation.
The PKC isozyme activated in this study is not an atypical PKC. There
are 11 isozymes in the PKC family, all requiring
phosphatidylserine (PS), a lipid found on the cytoplasmic side of
membranes, for activation. These isozymes are divided into three
subclasses based on their biochemical properties as follows:
conventional, isozymes requiring both DAG and calcium for
activation in addition to PS; novel, isozymes requiring DAG for
activation in addition to PS; and atypical (aPKC), isozymes requiring
only PS for activation (for review, see Newton, 1997). It is therefore
possible to distinguish between the subclasses based on their cofactor
requirement. The PKC activation that we described here was induced by
DAG surrogates, suggesting that the PKC investigated does not belong to
the aPKC subclass. Whether the kinase belongs to the conventional or
novel subclass remains to be determined.
PKC activators do not mimic light adaptation, nor do PKC inhibitors
prevent it
Exposure to bright light initiates a number of processes that
modulate the sensitivity of a photoreceptor. The term "light adaptation" generally refers to the rapid decline in sensitivity that
follows bright illumination. Light adaptation is usually accompanied by
a reduced response latency and duration (Fuortes and Hodgkin, 1964). In
Drosophila photoreceptors, the presence of an eye-specific
PKC appears to be necessary for light adaptation (Hardie et al., 1993).
Our pharmacological results do not support the concept that PKC
mediates light adaptation in Limulus ventral photoreceptors.
First, although ()-indolactam V decreases the cell's sensitivity to
light, it does not reduce response latency or duration (Fig.
2A; A. Dabdoub and R. Payne, unpublished
observations). Second, light adaptation desensitizes the cell's
response to InsP3 (Fein et al., 1984), whereas
()-indolactam V does not (Fig. 6B). Third, although
PKC inhibitors slightly sensitize the photoresponse and antagonize the
effect of PKC activators, they do not reverse the decrease in
sensitivity associated with light adaptation for the adaptation
protocols that we describe. Finally, injection of calcium ions alone
into the photoreceptor can fully mimic light adaptation (Brown and
Lisman, 1975; Fein and Charlton, 1977) and suppress the response to
InsP3 (Payne et al., 1986a) without any requirement for the addition of DAG or a surrogate. However, each of
these results is subject to caveats, and it is not possible to conclude
from these results that PKC plays no role in adaptation. In particular,
desensitization mediated by PKC could be a minor component of light
adaptation for the adaptation protocols that we chose. It may require
more prolonged or intense illumination, or it may be redundant to
calcium-mediated desensitization. It is also possible that the PKC
activated by light is not as susceptible to attack by PKC inhibitors as
that activated by ()-indolactam V. Calman et al. (1996) found that
the PKC inhibitors hypericin, chelerythrine, and calphostin C did not
inhibit PKC phosphorylation of endogenous substrates in
Limulus photoreceptor extracts, whereas the PKC peptide
inhibitor PKC 19-36 blocked phosphorylation. In addition, we found
incomplete reversal of PKC activation by the noncompetitive inhibitors
bisindolylmaleimide I and Gö 6976 (Fig. 3C),
indicating that there might be a component of the putative Limulus PKC activity that is resistant to the inhibitors used.
Site of action of PKC and its physiological role
We have physiologically localized the site of desensitization of
light-induced calcium release and depolarization by PKC activators to
an intermediate that is downstream from rhodopsin but upstream from
InsP3-induced calcium release. Therefore, we
propose that PKC activation results in the inhibition of the
phototransduction cascade at the level of the G-protein and PLC. One
possibility is that PKC might be directly phosphorylating the G-protein
and/or PLC in the cascade. PKC-mediated phosphorylation of PLC
isoforms has been proposed to decrease the activation of PLC by the
respective G-proteins (Ryu et al., 1990). Alternatively, PKC-mediated
phosphorylation of the G-protein's subunit may decrease its
activity (Aragay and Quick, 1999).
Injection of PKC activators into the lateral eyes of Limulus
induces structural changes leading to the internalization of microvillar membrane (Jinks et al., 1996), mimicking the natural shedding of microvillar membrane observed at dawn. Light-induced internalization of the microvillar membrane has also been observed in
isolated Limulus ventral nerve photoreceptors (Herman,
1991). The desensitization that we observe may therefore be a correlate or prelude to these anatomical changes. Another possibility is that PKC
activation may induce the translocation from the rhabdom of one of the
intermediates in the phosphoinositide cascade (Terakita et al., 1996),
resulting in a slow, long-term uncoupling of the phosphoinositide
cascade from rhodopsin that is distinct from the rapid calcium-mediated
reduction in sensitivity that is normally associated with light adaptation.
| |
FOOTNOTES |
Received March 31, 1999; revised Sept. 10, 1999; accepted Sept. 15, 1999.
This work was supported by National Institutes of Health Grant
EY-07743. We thank Dr. Ian Mather of the Department of Animal Sciences
(University of Maryland, College Park, MD) for the use of the confocal
microscope and Dr. Roger Hardie for his helpful criticism of this manuscript.
Correspondence should be addressed to Dr. Richard Payne, Department of
Biology, University of Maryland, College Park, MD 20742. E-mail:
rp12{at}umail.umd.edu.
| |
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ABSTRACT
INTRODUCTION
