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The Journal of Neuroscience, July 15, 1998, 18(14):5253-5263
Membrane Current Induced by Protein Kinase C Activators in
Rhabdomeric Photoreceptors: Implications for Visual Excitation
Maria del Pilar
Gomez and
Enrico
Nasi
Department of Physiology, Boston University School of Medicine,
Boston, Massachusetts 02118, and Marine Biological Laboratory, Woods
Hole, Massachusetts 02543
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ABSTRACT |
Visual excitation in rhabdomeric photoreceptors is thought to be
mediated by activation of a light-regulated phospholipase C (PLC) and
the consequent hydrolysis of phosphatidylinositol bisphosphate. Whereas
much attention has been devoted to inositol trisphosphate
(IP3) production and intracellular
Ca2+ release, little is known about the possible
role of the DAG branch in the generation of the light response. We have
tested the effect of chemically distinct surrogates of DAG on isolated
Lima photoreceptors. Application of the phorbol ester
PMA (0.5-10 µM) or the alkaloid ( )-indolactam (20-100
µM) from a holding potential of 50 mV elicited an
inward current, several hundred picoamperes in amplitude, accompanied
by a pronounced increase in membrane conductance. The stereoisomers
4 -PMA and (+)-indolactam were both inactive, arguing for the
specificity of the effects. Elevation of cytosolic Ca2+ by intracellular dialysis accelerated this
current, whereas chelerythrine antagonized it, suggesting the
involvement of PKC. The reversal potential of the membrane current
induced by PKC activators was approximately +10 mV; replacement of
extracellular Na with impermeant N-methyl-D-glucamine decreased its amplitude and
shifted the reversal potential in the negative direction. Stimulation
by PMA and ()-indolactam was accompanied by a pronounced depression
of light responsiveness; conversely, steady illumination reduced the
size of the current elicited by these PKC activators. Taken together,
these results support the notion that the DAG branch of the PLC
cascade, in addition to its suggested participation in visual
adaptation, may play a role in the activation of the photoresponse or a
component thereof, probably in synergy with IP3-mediated
Ca2+ release.
Key words:
visual excitation; PKC; diacylglycerol; rhabdomeric
photoreceptors; light-dependent conductance; calcium
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INTRODUCTION |
A detailed understanding of the
mechanisms by which photon absorption elicits a photocurrent in
rhabdomeric photoreceptors remains elusive, although substantial
evidence points to the critical involvement of a light-regulated
phospholipase C (PLC). Central pieces of information were the isolation
of a blind Drosophila mutant, norpA, (Pak et al.,
1969 ), together with the demonstration that this gene encodes a PLC
expressed in the retina (Bloomquist et al., 1988), and that both PLC
activity and light responses are rescued in norpA mutants
induced to express the norpA protein (McKay et al., 1995).
Light-induced hydrolysis of phosphatidylinositol bisphosphate
(PIP2) has been shown in squid (Baer and Saibil, 1988) and Drosophila (Devary et al., 1987), and most of the
research on photoexcitation mechanisms has focused on the resulting
production of inositol trisphosphate (IP3) (Szuts et
al., 1986; Brown et al., 1987) and the consequent release of
Ca2+ from internal stores (Brown and Rubin, 1984;
Payne et al., 1986b). However, despite the well documented excitatory
effects of both IP3 (Brown et al., 1984; Fein et al., 1984)
and Ca2+ (Payne et al., 1986a; Shin et al., 1993),
this branch of the PLC cascade alone has proved insufficient to fully
account for the photoresponse: (1) buffering intracellular calcium
changes slows the photocurrent in Limulus, but does not
abolish it (Lisman and Brown, 1975; Payne et al., 1986a); (2) low
molecular weight heparin, an inhibitor of IP3-induced
Ca2+ release, selectively reduces only the early
component of the photoresponse (Frank and Fein, 1991; Faddis and Brown,
1993); and (3) recent results indicate that a null mutation for the
allegedly sole isoform of IP3 receptor in
Drosophila eyes does not adversely affect the light response
(Acharya et al., 1997). An additional messenger substance, DAG, is
generated by PIP2 hydrolysis; the prime effector of DAG is
activation of PKC, which in turn phosphorylates a variety of target
proteins. In Drosophila, a null mutation for a
photoreceptor-specific PKC (inaC) (Smith et al., 1991) lacks normal deactivation of the light response (Smith et al., 1991; Hardie
et al., 1993) and also appears to suffer from a deficit in the light
adaptation process (Hardie et al., 1993). To date, however, the
possible involvement of PKC stimulation in the activation of any of the
membrane conductance mechanisms regulated by light has received
comparatively little attention. Nonetheless, some seminal observations
in this direction were briefly described by Brown et al. (1991). These
authors demonstrated that injection of the DAG surrogates into barnacle
photoreceptors caused a transient depolarization; the I-V
relationship of this response resembles that of the light-induced
current. We have used whole-cell clamp recording of membrane current
from isolated rhabdomeric photoreceptors of Lima scabra to
investigate the consequences of manipulating the DAG branch of the PLC
cascade on the membrane conductance, and we examined the interactions
with light stimulation and with alterations of the level of
intracellular calcium. A preliminary version of this report has been
presented in abstract form (Gomez and Nasi, 1997).
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MATERIALS AND METHODS |
Enzymatically dissociated rhabdomeric photoreceptors from
Lima scabra were obtained using the protocols described
previously (Nasi, 1991a). After plating in a recording flow chamber
mounted on the stage of an inverted microscope (ICM-405, Zeiss), the
photoreceptors were continuously superfused (1 ml/min) with artificial
seawater (ASW); a system of manifolds permitted the exchange of the
superfusate and was used to control the ionic composition of the bath.
To apply test substances extracellularly, we relied on a local
perfusion technique consisting of a puffer micropipette positioned in
the vicinity of the cell that could pressure eject a stream of test solution on activation of a solenoid-operated valve. Previous measurements obtained with a fluorescent dye included in the puffer pipette demonstrated that ~95% of solution exchange is attained within 200-400 msec, and that the ejection plume completely engulfs the region occupied by the target cell, as visualized with an image-intensified CCD camera (Gomez and Nasi, 1996a). Alternatively, in
some experiments, test compounds were administered intracellularly by
dialysis via the patch electrode.
Whole-cell patch-clamp recordings were performed as described
previously (Nasi, 1991b). All cell manipulations were performed under
dim near infrared illumination (>715 nm; Andover). Signals were
low-pass-filtered at 100 Hz for chemically activated currents and 1000 Hz for light-evoked currents, using a Bessel 4-pole filter. Records
were digitized on-line at a sampling rate of 200 Hz to 3 KHz.
Photostimulation was provided by a conventional optical rail delivering
small spots of light (~200 µm in diameter); the maximum effective
intensity corresponded to 58 × 1014
photons · sec 1 · cm2
at 500 nm, as measured with a calibrated radiometer (United Detector Technology, Hawthorne, CA). Neutral-density filters were interposed to
adjust incident light intensity. Voltage- and light-stimuli were
applied by a microprocessor-controlled programmable stimulator (Stim 6;
Ionoptix, Milton, MA).
Changes in cytosolic Ca2+ were detected using the
low-affinity fluorescent indicator calcium green 2 (Ca green 2;
Molecular Probes, Eugene, OR). The octapotassium salt of the probe was
dissolved in the intracellular solution filling the patch electrode at
a concentration of 65-100 µM. Excitation light was
provided by a 75 W xenon arc lamp (PTI, South Brunswick, NJ) filtered
by a dichroic reflector to reject wavelengths longer than 670 nm (Omega
Optical, Brattleboro, VT) and by an interference filter (480 nm, 40 nm bandwidth; Chroma Technology, Brattleboro, VT). The beam was brought to
the epi-illumination port of the microscope via a liquid light guide
(Oriel, Stratford, CT); the dichroic reflector in the microscope turret
had a cutoff wavelength of 505 nm. Emission light collected by a 100×,
1.3 NA oil-immersion objective (Nikon) was filtered sequentially by an
additional dichroic to reject  >610 nm and by a 535 nm interference
filter with a 50 nm bandwidth (all supplied by Chroma). An adjustable
rectangular mask (Nikon) located at a conjugated image plane was
positioned under infrared visualization to restrict the collection of
fluorescence emission to the region of interest (usually the
light-transducing rhabdomeric lobe of an isolated photoreceptor) to
minimize background light. The fluorescence signal was detected by a
photomultiplier tube (R4220 PHA; Hammamatsu, Bridgewater, NJ) operated
at 800 V in photon-counting mode using a window discriminator and a
rate meter (F-100T and PRM-100, respectively; Advanced Research
Instruments, Boulder, CO). An analog voltage proportional to the counts
accumulated in bins of programmable duration (typically,
103 sec) was fed to the analog-to-digital
interface of the computer.
Solutions. ASW contained (in mM): 480 NaCl, 10 KCl, 10 CaCl2, 49 MgCl2, 10 HEPES, and 5.5 glucose, pH 7.8 (NaOH).
N-methyl-D-glucamine (NMDG) replaced sodium in
Na-free seawater, whereas in low-Ca2+ ASW (250 µM) and in nominally Ca-free artificial seawater (0-Ca ASW), calcium was replaced with magnesium on an equimolar basis. A
solution lacking Ca2+ and with Mg reduced to 1 mM was also used; in this case, the concentration of NaCl
was increased to 567 mM. Phorbol 12-myristate 13-acetate
(PMA), 4-PMA, (+)-indolactam, and chelerythrine were purchased from
Alexis Corporation/LC Laboratories (Woburn, MA). ()-Indolactam V was
obtained both from Alexis and from Calbiochem (San Diego, CA). These
substances were dissolved in DMSO at 10-50 mM, aliquoted,
and kept at 75°C. Synthetic inositol 1,4,5 trisphosphate (IP3) was purchased from Alexis, dissolved in a 10 mM stock solution, aliquoted, and kept at 75°C.
The standard intracellular solution used to fill whole-cell
micropipettes contained 200 mM K-glutamate, 100 mM KCl, 22 mM NaCl, 5 mM Mg ATP, 10 mM HEPES, 1 mM EGTA, 100 µM GTP,
and 300 mM sucrose, pH 7.3. The internal solution with
elevated Ca2+ had a similar composition except that
0.8 mM CaCl2 was added to it, yielding an
estimated free calcium concentration of 1 µM, according to the program Chelator (courtesy of Dr. Theo
Shoenmakers, University of Nijmegen, The Netherlands).
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RESULTS |
PKC activators stimulate a membrane conductance
PMA, one of the most potent phorbol ester activators of PKC (for
review, see Nishizuka, 1984), was first tested by local superfusion. Figure 1 shows the effect of pressure
ejecting PMA at a concentration of 1 µM from a
micropipette located ~50 µm from a cell. The photoreceptor was
maintained in darkness and voltage clamped at 50 mV. Repetitive rectangular voltage steps (3 mV peak to peak) were superimposed on the
holding potential to monitor changes in membrane resistance. After
several seconds of application of PMA, an inward current gradually
developed, reaching an amplitude of ~360 pA. Concomitantly, the size
of the current steps elicited by the voltage pulses grew (Fig. 1,
insets), indicating a conductance increase from a basal value of
1.6-18.5 nS (an increment of >620%). All cells tested in this manner
exhibited a similar response. At a holding potential of 50 mV, the
mean amplitude of the inward current was 391 ± 102 pA (±SD;
n = 6); the conductance of the membrane increased by
18.2 ± 7.2 nS. The latency of the response, measured as the time
required to reach 10% of the maximum inward current
(t10%), was 61 ± 32 sec. Little
recovery could be observed after interrupting delivery of the PMA, at
least within the useful temporal window for recording (usually
20-30 min). This limitation, unfortunately, precluded the possibility
of repeated tests on a given cell.
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Figure 1.
Activation of a membrane current by PMA.
A, A photoreceptor cell was voltage clamped at 50 mV,
with a repetitive 3 mV rectangular pulse superimposed on the holding
potential. A puffer pipette containing 1 µM PMA in ASW
(final DMSO concentration 0.1%) was positioned ~50 µm away from
the cell. The thick bar indicates pressure ejection. A
large inward current was elicited. The change in the current steps
(insets) indicates that an increase of membrane
conductance occurred. B, Lack of effects of dialysis
with normal internal solution (top trace),
pressure-application of ASW, ASW containing 5% DMSO, and 1 µM 4-PMA. In the last trace, no voltage
command steps were superimposed on the holding potential; the downward
deflections are quantum bumps. Calibration: 1 min. C,
Inward current elicited by internal dialysis with 0.5 µM
PMA via the patch pipette. A similar inward current, as in
A, was elicited, but with a reduced latency.
Insets illustrate the increase in conductance during the
development of the inward current.
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In vitro, PMA has been shown to be effective as a PKC
activator at nanomolar concentrations (Nishizuka, 1984); because of the
mode of application we used, it is difficult to gauge the amount of PMA
reaching its target sites in the interior of the cell. Nevertheless, it
appears that the responses shown above were nearly saturating, because
similar tests conducted with 10 µM PMA produced only
marginally larger currents (average amplitude 536 ± 90 pA;
n = 3). This value was not statistically significantly different from that obtained with 1 µM.
The specificity of the effects of PMA were assessed in several ways, as
shown in Figure 1B. The top trace in
Figure 1B was simply recorded in the dark with a
standard intracellular solution immediately after gaining access to the
cell interior and confirms that in the absence of additional
manipulations the holding current remains stable. To rule out possible
confounding effects of the puffer that may stem from the mechanical
strain induced by the turbulence during pressure ejection, control
cells were tested with a puffer pipette containing ASW; ejection of a
stream of solution onto these photoreceptors produced no detectable
change in holding current (n = 7) (Fig
1B, second trace). Because PMA had to be
dissolved initially in DMSO, we also examined possible effects of this
solvent. Control measurements were therefore performed by pressure
ejecting ASW containing DMSO at concentrations of 1%, 2%, and 5%
(n = 2, 2, and 5, respectively) onto cells voltage clamped at 50 mV in the dark. As shown in Figure
1B, third trace, the holding current was
unaffected by this treatment, although the DMSO concentration was 5- to
10-fold higher than in the test solutions containing PMA (typically
<0.5% 1%). Finally, we conducted similar experiments using the
4 isomer of PMA, which is nearly inert and thus serves as a good
negative control. Figure 1B, bottom trace,
demonstrates that application of 4-PMA at 1 µM
produced virtually no change in membrane current (average 21 ± 11 pA; n = 4). We also tested the effect of direct
intracellular application of PMA instead of puffing. To this end, PMA
was included in the internal solution (0.5-1 µM) and
dialyzed through the patch microelectrode. Previous measurements using
the same cells demonstrated that small molecules in the pipette-filling
solution equilibrate with the cytosolic compartment of the rhabdomeric
lobe and in fact reach the sites where light-dependent ion channels are
located (Gomez and Nasi, 1996a). Figure 1C shows that
shortly after gaining access to the cell interior, a large inward
current was elicited; the size of the responses obtained (mean
amplitude 277 ± 123 pA; n = 3) was no larger than
those produced by extracellular administration, but the time necessary
to reach 10% of the final amplitude was significantly shorter (over
fivefold briefer) (11.9 ± 6 sec; n = 3), with
virtually no latent period. Intracellular application of 1% DMSO
without PMA was ineffective.
To support the notion that the observed effects of phorbol esters
indeed result from an interaction with the DAG branch of the PLC
cascade, we examined the effects of a chemically distinct substance
that has a similar stimulatory action on PKC. Indolactam V is an
alkaloid activator of PKC, presumed to bind at the same site on the
enzyme at which phorbol esters act (Heikkilä and Åkerman, 1989).
Figure 2A shows an
experiment in which ()-indolactam, the active stereoisomer, was
applied in the dark by ejection from a puffer pipette positioned near a
photoreceptor cell voltage clamped at 50 mV. The concentration of
indolactam was 100 µM; a square wave perturbation of the
command voltage (3 mV) was used to monitor changes in membrane
conductance. Several seconds after activating the puffer pipette, an
inward current developed (peak amplitude ~400 pA), accompanied by a
conspicuous increase in membrane conductance. Similar effects were
observed in all seven photoreceptors stimulated with 100 µM ()-indolactam; the mean amplitude of the response
was 415 ± 54 pA, and the observed latency was 31.4 ± 14 sec. To test for specificity of the effects of indolactam, control
experiments were performed with the mirror-image molecule (+)-indolactam; this stereoisomer is relatively inert and is
~100-fold weaker in competing with PMA in binding assays (Fujiki et
al., 1984) and in activating PKC-mediated processes (Heikkilä and Åkerman, 1989). Figure 2A, bottom trace,
shows that extracellular administration of (+)-indolactam at the same
concentration of 100 µM failed to induce any change in
membrane current or in membrane conductance (n = 3). We
also examined the effect of varying the concentration of
()-indolactam in the range between 5 and 100 µM.
Figure 2B summarizes the obtained data pooled from 22 cells (n = 5-7 in each group). The amplitude of the
membrane current and the conductance changes appear to reach saturation
at concentrations <50 µM; the latency of the response
(t10%) also displayed a marked dose
dependency (Fig. 2C), becoming shorter at higher concentrations, which is an effect that probably reflects in part the
increased rate of entry of the compound into the cell. Overall, all of
the 22 photoreceptors examined under these conditions and stimulated
with different concentrations of indolactam at 50 mV responded with
an inward current of variable amplitude and, whenever measured, the
membrane conductance increased. This compound was also effective when
applied intracellularly. As shown in Figure 2D,
dialysis of 50 µM ()-indolactam V evoked inward
currents of similar characteristics; the amplitude was not larger than that measured with local superfusion (average 338 ± 235 pA;
n = 4; mean increase in membrane conductance 19.6 ± 12 nS), but the latencies were dramatically reduced
(t10% 4.0 ± 0.9 sec).
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Figure 2.
Effect of indolactam V on membrane current.
A, Local superfusion with 100 µM
()-indolactam of a rhabdomeric cell held at 50 mV in the dark
caused a large inward current accompanied by an increase in membrane
conductance. Administration of the stereoisomer (+)-indolactam at the
same concentration was ineffective (bottom trace).
B, Effects of different concentrations of indolactam on
the peak amplitude of the inward current elicited at 50 mV
(filled squares) and membrane conductance
(open squares). Error bars indicate SD. The response
appears to saturate at <50 µM. C, Time
required to reach 10% of the maximum amplitude of the current, plotted
as a function of concentration. D, Inward current
obtained by administering 50 µM ()-indolactam
intracellularly. Notice the dramatic reduction in the latency of the
inward current.
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To determine whether the membrane currents observed with administration
of these DAG surrogates indeed involved activation of PKC, we examined
the effect of treating cells with an inhibitor of PKC on the response
to indolactam. Chelerythrine is a highly specific PKC antagonist with
half-maximal effectiveness in the submicromolar range and a >100-fold
selectivity for PKC over either PKA or Ca-CaM PK (Herbert et al. 1990).
To assess the ability of chelerythrine to interfere with the response
elicited by PKC activators, we first dialyzed ()-indolactam
internally, causing the development of an inward current at
Vh 50 mV, and then applied 50 µM
chelerythrine with a puffer pipette. As shown in Figure 3, pressure ejection of chelerythrine
reduced the inward current and caused the membrane conductance to
decrease toward the basal level. A similar effect was obtained in three
of four photoreceptors tested in this manner. The average reduction in
the indolactam-induced inward current was 49 ± 27%. In the
absence of indolactam, application of chelerythrine did not affect the
holding current (Fig. 3, bottom trace) and appeared not to
depress the peak amplitude of the photocurrent (data not shown).
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Figure 3.
DAG surrogates act via PKC activation. To
ascertain whether the current induced by administration of indolactam
is mediated by its ability to activate PKC, photoreceptors were first
stimulated with 50 µM ()-indolactam V applied by
internal dialysis. Once a conspicuous inward current developed at 50
mV, a puffer pipette was used to pressure eject 50 µM of
the PKC inhibitor chelerythrine. After several seconds of application
of the antagonist, the inward current and the associated increase in
membrane conductance were substantially reversed. Bottom
trace shows that application of chelerythrine to a control cell
dialyzed with the normal internal solution was ineffective.
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Interaction of PKC activators with calcium and with
light stimulation
Activation of PKC by DAG is profoundly facilitated by the presence
of elevated levels of cytosolic calcium (Nishizuka, 1984). We examined
the effects of internally dialyzing photoreceptors with a solution
containing 1 µM free Ca before external application of
PKC activators. Figure
4A shows typical traces
obtained by puffing 5 µM indolactam in the dark, using
either the standard Ca2+-free internal solution
(Fig. 4, ) or one containing elevated Ca2+ (Fig.
4, +). In the presence of high Ca2+, the response
amplitude was significantly enhanced (plateau not shown in Fig. 4), but
the most dramatic effect was an acceleration of the action of
indolactam: compared with the Ca2+-free
intracellular solution, the latency decreased from 225 ± 35 sec
(n = 5) to 72 ± 29.7 sec (n = 6).
Figure 4B, top, shows the membrane
currents from four different cells internally perfused with the high
Ca2+ solution and stimulated with a higher
concentration of indolactam (50 µM). In this case, the
latency was 9.3 ± 10.7 sec (n = 8) compared with
61 ± 36 sec for 0 Cai (n = 7), but
the elevated Ca2+ did not increase the steady-state
size of the response, probably because of saturation. Control
measurements using the high Ca2+ internal solution
without indolactam (Fig. 4B, bottom)
failed to reveal any development of a net macroscopic current and only induced a progressive increase in membrane noise. A two-factor ANOVA
indicated that the effects of internal calcium on response latency were
statistically highly significant (p < 0.01),
and so was the interaction of indolactam × Ca2+ (p < 0.01). A
qualitatively similar though less pronounced effect of
Ca2+ was observed with PMA, although the comparison
was only done at a single saturating concentration (10 µM): elevated internal Ca2+ decreased
the latency by 38% (n = 3 in each condition).
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Figure 4.
Facilitation of the effects of PKC activators by
elevation of intracellular Ca2+. A,
Recordings were obtained from two different photoreceptor cells
internally dialyzed with Ca-free solution () and with 1 µM free calcium (+), respectively. The inward current
evoked by local application of 5 µM indolactam
(top thick bar) had a much more rapid onset and a larger
amplitude when an internal solution with elevated calcium was used.
B, Responses elicited by 50 µM indolactam
in four different cells dialyzed with high Ca2+. In
all cases the inward current attained its peak amplitude within 1 min.
Bottom traces are from four control cells dialyzed with
1 µM free calcium without indolactam application.
C, Mean latency (t10%) of the inward
current evoked by the two concentrations of indolactam in the presence
or absence of Ca2+ in the internal solution. Error
bars indicate the SD.
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The physiological relevance of the above observation is that in other
microvillar photoreceptors a pronounced increase in cytosolic calcium
accompanies the light response (Brown and Blinks, 1974). We ascertained
that a similar phenomenon occurs in Lima rhabdomeric
photoreceptors. Figure 5A
shows the simultaneous recording of membrane current and fluorescence
from a 5 × 5 µm window encompassing only the rhabdomeric lobe
of an isolated photoreceptor that was loaded with Ca green 2 via the
patch electrode and voltage clamped at 50 mV. The optical signal
jumped abruptly at the onset of the excitation light, and after a
latency of ~30 msec, a marked increase in fluorescence occurred,
coincident with the activation of the saturating photocurrent. Similar
results were obtained in four additional cells. Control measurements in
which the excitation light was presented under light-adapted conditions
(i.e., shortly after another stimulus) failed to evoke a light
response, and the fluorescence signal only exhibited a square
transition (data not shown). Conversely, the use of Ca-insensitive
fluorescent compounds, such as fluorescein, also resulted in a square
optical signal, although a robust photocurrent was elicited
(n = 3). These observations provide assurance that the
kinetics of the Ca green 2 signal recorded in dark-adapted cells indeed
reflect a light-induced increase in cytosolic Ca2+,
concomitant with the visual excitatory response. Figure 5B
shows a similar recording obtained from a photoreceptor bathed for 8 min in 0-Ca ASW; a similar Ca2+ rise accompanies the
light response (n = 3). The same result was also
obtained during superfusion with 0 Ca2+/1
mM Mg2+ solution (n = 2). These observations indicate that the photo-induced Ca2+ increase is attributable to, at least in part,
release from internal stores.
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Figure 5.
Light-induced Ca2+ release and
effects of IP3. A, Left,
Simultaneous recording of membrane current (top trace)
and fluorescence (bottom trace) in a photoreceptor
loaded with 100 µM Ca Green 2 and voltage-clamped at 50
mV. At the onset of the excitation light step (500 msec, 480 nm), the
optical signal jumped abruptly to a baseline level, and ~30 msec
later a secondary increase in fluorescence was observed, reflecting the
rise in cytosolic calcium. Right, A similar result was
obtained in a cell bathed for many minutes in a Ca-free solution.
B, Light-intensity series obtained with the standard
intracellular solution or one containing 10 µM
IP3, respectively; access to the cell interior was
through the somatic lobe. The normalized peak amplitude of the
photocurrent plotted as a function of light intensity
(bottom) reveals a marked desensitization induced by
IP3. Calibration: 800 pA, 400 msec. C,
Oscillatory inward current evoked by dialysis with IP3
directly into the rhabdomeric lobe of a photoreceptor. The recording
started immediately on rupturing the patch of membrane to gain access
to the cell interior. D, Left, Comparison
of the current elicited in the dark by puffer application of 50 µM ()-indolactam (horizontal bar) in a
cell internally dialyzed with the standard intracellular solution and
one dialyzed with 10 µM IP3.
Right, Histogram comparing the mean latency
(t10%) of the indolactam-evoked current in control
and IP3-treated photoreceptors (n = 7 and 9, respectively).
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The conclusion that the Ca2+ elevation results from
intracellular release suggests that, like in other invertebrate
photoreceptors, light may mobilize IP3; in
Limulus, IP3 rapidly elevates
Ca2+, leading to desensitization of the light
response, and if injected into the rhabdomeric lobe, it also evokes
bursts of inward current (for review, see Payne et al., 1988). Figure
5B shows intensity series obtained in a control cell and one
dialyzed with 10 µM IP3 into the soma. In the
latter, the light response was significantly depressed; the plot in
Figure 5B, bottom, shows that sensitivity shifted
by ~1.35 log. A similar reduction of responsiveness was observed in
four additional photoreceptors. Internal perfusion through the villous
rhabdomeric lobe proved challenging; nevertheless, Figure 5C
shows a successful instance in which bursts of inward current, often
exceeding 80 pA in amplitude, occurred as soon as the patch of membrane
was ruptured. These results indicate that IP3 is likely to
play a similar role in Lima as in other rhabdomeric
photoreceptors. We then compared the effect of IP3 on the
conductance induced by application of ()-indolactam. Figure 5D shows the membrane current evoked in the dark by puffing
50 µM indolactam in a control cell and in one dialyzed
with 10 µM IP3. IP3 significantly
shortened the latency of the response to indolactam. Pooling the data
from several cells in each condition (Fig. 5D) showed that
the effect qualitatively resembles that of 1 µM
intracellular Ca2+ (Fig. 4C), although
less pronounced.
Although the results presented above indicate that application of PKC
activators reliably induces a membrane conductance in rhabdomeric
photoreceptor cells, a central question concerns a possible
relationship of these effects to the visual excitation process. We
took several approaches to examine this issue. To the extent that
PKC activators tap an effector mechanism that is also involved in the
generation of the photocurrent, one would expect to find an interaction
between application of these chemical agents and the effects of
photostimulation. We first tested this proposition by comparing the
effects of light flashes before and after the induction of a current by
either indolactam or PMA. Figure
6A shows typical
photoresponses obtained under voltage clamp at 50 mV. Under control
conditions (Fig. 6A, bottom trace), the
peak amplitude of the near-saturating current was several nanoamperes;
after comparable responses were repetitively obtained, indolactam (20 µM) was locally applied in the dark, inducing an inward
current of ~500 pA (data not shown). Subsequent presentation of
another identical flash only evoked a much reduced photoresponse (Fig.
6A, top trace). In all photoreceptors
exposed to indolactam under various conditions (n > 20), a marked depression of the light response was observed; the
reduction in light sensitivity, measured as the intensity required to
elicit a criterion response amplitude, was ~1.5 log. A comparable
effect was also observed in cells stimulated with PMA, either
extracellularly (1 µM; n = 4) or by
internal dialysis (0.5 µM; n = 3).
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Figure 6.
Interactions between light and PKC activators.
A, Depression of the photocurrents by application of
()-indolactam (50 µM). A photoreceptor cell was voltage
clamped at 50 mV and stimulated with 100 msec flashes of light
(3.7 × 1014
photons · sec1 · cm2);
subsequently, indolactam was applied by a puffer pipette, evoking a
sustained inward current (data not shown). Delivery of another
identical flash (top trace) elicited a dramatically
reduced photocurrent. B, Effect of steady light on the
amplitude and latency of the response to indolactam. The recordings at
the top illustrate the responses evoked by 50 µM indolactam (thick lines) in two cells
maintained in the dark (left) and two cells continuously
illuminated (3.7 × 1014
photons · sec1 · cm2).
Bottom shows the mean steady-state current amplitude
(filled bars) and the mean latency
(t10%) (open bars)
pooled for six cells in each of the two conditions. Error bars indicate
SD.
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A complementary approach entails comparing the effects of indolactam
application in the dark versus during sustained illumination. In the
latter case, the light was turned on 2-3 min before the beginning of
the recording to ensure a near steady-state of the light response and
to minimize any slow drift in membrane current that may result from
further light adaptation. Figure 6B, top traces, shows responses elicited by puffing 50 µM
indolactam, either in the dark or during steady illumination (in
different cells). Figure 6B, bottom, shows
average data for several cells tested in each of the two conditions.
The response amplitude (Fig. 6B, filled
bars) was reduced nearly threefold in the presence of light [from
594 ± 208 pA (n = 7) to 198 ± 89 pA
(n = 6); p < 0.02 by the Wilcoxon
test] (Hollander and Wolfe, 1973). It is also worth noting that during
light the response latency (29 ± 3.3 sec; n = 6)
was ~57% shorter than in darkness (67 ± 41 sec), this
difference being statistically significant at p = 0.05. Considering that in rhabdomeric cells photostimulation
triggers an elevation of cytosolic Ca2+ (Brown and
Blinks, 1974), the observed acceleration is in agreement with the
results described above and illustrated in Figure 4 in which
photoreceptors were intracellularly dialyzed with solutions containing
a high Ca2+ concentration before the administration
of PKC activators.
Ionic basis of the current induced by PMA and indolactam
The conduction and selectivity properties of the current evoked by
PKC activators were briefly examined. Figure
7A, top,
illustrates the effect of omitting extracellular sodium: replacement of
Na with NMDG resulted in a significantly reduced amplitude of the inward current evoked by application of 50 µM indolactam
at Vm = 50 mV. Figure 7A,
bottom, summarizes the data obtained from three cells tested
in each condition; the average peak current in ASW was 632 ± 32 pA, whereas that in 0 Na was 114 ± 49 pA. As shown in Figure
7B, we examined the effect of Na removal on the reversal
voltage of the current evoked by PKC activators. Figure 7B,
top traces, shows the effect of puffing ()-indolactam in
ASW at two holding voltages (in different cells); at +30 mV a
conspicuous outward current was elicited (n = 3),
whereas at +10 mV a minute inward current is still detectable. Similar
results were obtained with PMA (n = 3: 2 cells tested
at +30, 1 at +10 mV). The large size of the outward current at a
holding potential only ~20 mV positive of Vrev
suggests a pronounced outward rectification (compare with the average
current at 50 mV shown in Fig. 7A). Figure 7B,
bottom, shows that replacement of extracellular sodium with
NMDG markedly affected the reversal potential: at 0 mV the current
elicited by indolactam is already outwardly directed (n = 3). Testing at 30 mV failed to reveal a consistent response (Fig.
7B, bottom, bottom trace), whereas at
voltages more negative than 40 mV the response was inward (data not
shown; n = 4). Exact determination of the reversal
voltage is difficult, because each test must be performed in a
different cell so that variability in responsiveness across
photoreceptors precludes the use of interpolation. Nevertheless, one
can define the range in which responses invert polarity and estimate an
approximate shift of Vrev after Na removal on
the order of 40 mV. The observation that in Na-free conditions an
inward current can be evoked at membrane potentials substantially more
positive than EK indicates the participation of
other ions; calcium is an obvious candidate. Interestingly, when the
extracellular Ca2+ concentration in ASW is reduced
to 250 µM, an inward response is evoked by puffing PMA,
but its peak amplitude (mean 237 ± 38 pA; n = 3)
was significantly less than in normal ASW (p < 0.05, one-sided Wilcoxon rank-sum test).
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Figure 7.
Effect of external sodium removal on the current
induced by PKC activators. A, Top,
Replacement of Na with NMDG reduced the amplitude of the inward current
evoked by application of 50 µM indolactam (thick
horizontal lines). Membrane potential: 50 mV. Calibration:
100 pA, 1 min. Bottom, pooled data for several cells
stimulated in the presence and absence of Na (n = 3 in each condition). Error bars indicate SD. B,
Top, Reversal of the indolactam-evoked current in ASW.
The membrane potential was clamped at the levels indicated; the traces
were recorded from different cells. Bottom, Shift in the
reversal voltage during superfusion with Na-free solution (replaced
with NMDG).
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DISCUSSION |
The present results demonstrate that structurally unrelated DAG
surrogates applied to rhabdomeric Lima photoreceptors induce a marked increase in membrane conductance and an ionic current, which,
at a holding voltage near the cells' resting potential, is inwardly
directed. Similar excitatory effects are obtained with extracellular
local perfusion or with intracellular dialysis of such compounds, but
in the latter case the onset of the current is significantly more
rapid. The ineffectiveness of a variety of control treatments,
including the administration of inert stereoisomers of both indolactam
and of PMA, argues for specificity of these effects. The relative
potency of PMA and ()-indolactam V for inducing an increase in
membrane conductance in rhabdomeric photoreceptors mirrors their
differential effectiveness as activators of protein kinase C in
in vitro assays (Heikkilä and Åkerman, 1989).
Moreover, the membrane current elicited by indolactam could be
partially reversed by administration of chelerithrine, suggesting that
the observed effects are indeed mediated by activation of a PKC.
Elevation of intracellular calcium, which in vivo acts in
synergy with DAG to activate PKC, produced a strong facilitation of the
effect of both indolactam and PMA by dramatically shortening the
latency of the evoked current. Administration of IP3 also
accelerates the onset of the response to indolactam. Because light
stimulation leads to an increase in
[Ca2+]i in the rhabdomeric lobe, which
is attributable to release from internal stores (Fig. 5), the
modulatory effect of calcium is likely to be physiologically
relevant.
Light responsiveness of the photoreceptors is significantly decreased
after stimulation with PKC activators; this effect is in line with the
proposition that PKC plays an important role in light adaptation, as
suggested from studies with PKC-deficient mutant flies (Hardie et al.,
1993). However, the converse effect was also obtained; that is to say,
the indolactam-evoked membrane currents are reduced when this substance
is applied during illumination. The latter finding suggests that in
addition to desensitization, some mutually occlusive action of these
two stimulatory treatments exists with respect to activation of
membrane ion channels. This observation is compatible with the notion
that PKC activators also tap some effector mechanism that is controlled
by light stimulation. Several characteristics of the current evoked
by PKC activators are indeed remindful of the light response. Its
reversal potential (Erev) is several
millivolts positive of 0 mV and is displaced in the negative direction
after replacing extracellular Na with impermeant NMDG; the magnitude of
this shift, a few tens of millivolts, is reminiscent of that of the
photocurrent ( Erev = ~47 mV; Gomez and
Nasi, 1996a), indicating that a sizable portion of the PCK activator-induced current is carried by sodium ions. Furthermore, the
large outward currents measured at +30 mV indicate a substantial rectification in the outward direction.
A pertinent question, therefore, concerns a possible scheme that
encompasses a role for DAG in the visual excitation process, in
addition to the well documented involvement of IP3 and
Ca2+. One suggestion is inspired by work conducted
by Payne and Fein (1986) in Limulus ventral photoreceptors.
These authors examined the leading edge of the photocurrent and
demonstrated a marked supralinearity of its rate of rise as a function
of the density of impinging photons; this acceleration was antagonized
by Ca buffers. They proposed a model in which two parallel cascades are
triggered by light, one that controls light-dependent channels and the
other that accelerates the rate of the first, and suggested that
the accelerating agent could be calcium. Faddis and Brown (1993)
examined the effects of intracellular injection of heparin and BAPTA
into Limulus ventral photoreceptors and also concluded that
visual excitation can proceed in the absence of IP3-induced increases in [Ca2+]i, albeit
with a reduction in gain, speed of transduction, and adaptation. It
would be tempting to speculate that the DAG branch of the
light-triggered cascade could control light-dependent membrane conductance changes, whereas the IP3 branch would modulate
the gain and speed of the response by providing the appropriate
increase in enzymatic reaction rates via Ca2+.
In principle, the present results are compatible with such a view,
whose appeal is obvious. However, at least in its simplest form, this
scheme presents shortcomings that deserve closer scrutiny: although the
maximum size of the current elicited by PKC activators in
Lima is substantial (hundreds of picoamperes), it is still considerably smaller than the peak-amplitude of the photocurrent evoked
by a flash of saturating intensity (several nanoamperes). One
possibility that could account for the quantitative discrepancy in the
effectiveness of the two types of stimulation (under the assumption
that light and PKC activators indeed converge onto a common target
mechanism) is that whereas light is delivered in a nearly instantaneous
manner, chemicals applied either by superfusion or by intracellular
dialysis require tens of seconds to reach their target sites, and their
local concentration rises only gradually. It is conceivable that such
slow onset of the stimulus allows for adaptation to set in in such a
way that it is not possible to observe the full-blown response.
Alternatively, the current activated by DAG surrogates may only be a
component of the whole photoresponse. Over the last few years, evidence has accumulated for the existence of multiple light-dependent conductance mechanisms that contribute to the photocurrent in rhabdomeric photoreceptors of several species, including
Lima (Nasi, 1991b), Limulus (Deckert et al.,
1992), Drosophila (Hardie and Minke, 1992), and
Hermissenda (Detwiler, 1976). The relative contribution of
the various components to the total photocurrent is quite
heterogeneous, and in Lima the early transient is typically several-fold larger than the slower "tail," although the extent of
temporal overlap precludes an exact estimate. It is conceivable that
PKC activators interact with the photocurrent-generating machinery,
triggering the smaller component. This possibility is plausible because
if DAG and PKC controlled the main component of the light response, PKC
inhibitors would be predicted to exert a strong overall antagonistic
effect; by contrast, our initial measurements with chelerythrine failed
to consistently show a decrease of the peak amplitude of the light
response. This result is also in line with the observation that in
inaC mutants the size of the photocurrent does not appear to
be systematically lower than in wild type (Smith et al., 1991; Hardie
et al., 1993). Another possibility is that the target of PKC activators
is the slow light-evoked inward current that we have recently described in Lima (Gomez and Nasi, 1996b); this conductance develops
in response to bright lights a few seconds after the termination of the
rapid complex photoresponse. Its sluggish time course, requirement for
saturating levels of stimulation, and calcium permeability are
reminiscent of certain Ca-depletion activated currents found in many
cells that use the IP3 signaling pathway (for review, see
Fasolato et al., 1994). In other systems, such as Xenopus
oocytes and insulin-secreting cell lines, it has in fact been suggested
that PKC activation is intimately involved in the control of such ionic
mechanisms (Bode and Göke, 1994; Petersen and Berridge,
1994).
For now, positive identification of the conductance elicited by PKC
activators in Lima photoreceptors with a specific
light-dependent ionic mechanism remains challenging, because their
respective reversal voltages differ only by several millivolts, and our
measurements presently lack the sensitivity required to make such fine
distinctions. Moreover, ionic manipulations are poor tools to help
discriminate among relatively unselective channels, and no
pharmacological tools are yet available to dissect them. In any case,
the present observations are strongly suggestive of an involvement of
the diacylglycerol branch of the light-triggered PLC cascade in the control of ion channels and could lead to a clarification of some of
the persisting complexities concerning the generation of the light
response in rhabdomeric cells.
| |
FOOTNOTES |
Received Jan. 15, 1998; revised April 30, 1998; accepted May 4, 1998.
This work was supported by National Institutes of Health Grant RO1
07559.
Correspondence should be addressed to Dr. Maria del Pilar Gomez,
Department of Physiology, Boston University School of Medicine, 80 East
Concord Street, Boston, MA, 02118.
| |
REFERENCES |
-
Acharya JK,
Jalink K,
Hardy RW,
Hartenstein V,
Zuker CS
(1997)
InsP3 receptor is essential for growth and differentiation but not for vision in Drosophila.
Neuron
18:881-887[Web of Science][Medline].
-
Baer KM,
Saibil HR
(1988)
Light- and GTP-activated hydrolysis of phosphatidylinositol bisphosphate in squid photoreceptor membranes.
J Biol Chem
263:17-20[Abstract/Free Full Text].
-
Bloomquist BT,
Shortridge RD,
Schneuwly S,
Perdew M,
Montell C,
Steller H,
Rubin G,
Pak WL
(1988)
Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.
Cell
54:723-733[Web of Science][Medline].
-
Bode H-P,
Göke B
(1994)
Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F.
FEBS Lett
339:307-311[Web of Science][Medline].
-
Brown JE,
Blinks JR
(1974)
Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors.
J Gen Physiol
64:643-665[Abstract/Free Full Text].
-
Brown JE,
Watkins DC,
Malbon CC
(1987)
Light-induced changes in the content of inositol phosphate in squid (Loligo pealei) retina.
Biochem J
247:293-297[Web of Science][Medline].
-
Brown JE,
Rubin LJ
(1984)
A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors.
Biochem Biophys Res Commun
125:1137-1142[Web of Science][Medline].
-
Brown JE,
Rubin LJ,
Ghalayni AJ,
Tarver AP,
Irvine RF,
Berridge MJ,
Anderson RE
(1984)
Myo-inositol polyphosphate may be a messenger for visual excitation in Limulus photoreceptors.
Nature
311:160-163[Medline].
-
Brown HM,
Burnham J,
Smolley J
(1991)
Diacylglycerol surrogates induce membrane currents in Balanus photoreceptors [abstract].
Biophys J
59:530.
-
Deckert A,
Nagy K,
Helrich CS,
Stieve H
(1992)
Three components in the light-induced current of the Limulus ventral photoreceptor.
J Physiol (Lond)
453:69-96[Abstract/Free Full Text].
-
Detwiler PB
(1976)
Multiple light-evoked conductance changes in the photoreceptors of Hermissenda crassicornis.
J Physiol (Lond)
256:691-708[Abstract/Free Full Text].
-
Devary O,
Heichal O,
Blumenfeld A,
Cassel D,
Suss E,
Barash S,
Rubinstein CT,
Minke B,
Selinger Z
(1987)
Coupling of photoexcited rhodopsin to inositol phospholipid hydrolysis in fly photoreceptors.
Proc Natl Acad Sci USA
84:6939-6943[Abstract/Free Full Text].
-
Faddis MN,
Brown JE
(1993)
Intracellular injection of heparin and polyamines. Effects on phototransduction in Limulus ventral photoreceptors.
J Gen Physiol
101:909-931[Abstract/Free Full Text].
-
Fasolato C,
Innocenti B,
Pozzan T
(1994)
Receptor-activated Ca2+ influx: how many mechanisms for how many channels?
Trends Pharmacol Sci
15:77-83[Medline].
-
Fein A,
Payne R,
Corson WD,
Berridge MJ,
Irvine RF
(1984)
Photoreceptor excitation and adaptation by inositol 1,4,5-trisphosphate.
Nature
311:157-160[Medline].
-
Frank TM,
Fein A
(1991)
The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors.
J Gen Physiol
97:697-723[Abstract/Free Full Text].
-
Fujiki H,
Suganuma M,
Nakayasu M,
Tahira T,
Endo Y,
Shudo K,
Sugimura T
(1984)
Structure-activity studies on synthetic analogs (indolactams) of the tumor-promoter teleocidin.
Gann
75:866-870[Web of Science][Medline].
-
Gomez M,
Nasi E
(1996a)
Ion permeation through light-activated channels in rhabdomeric photoreceptors: role of divalent cations.
J Gen Physiol
107:715-730[Abstract/Free Full Text].
-
Gomez M,
Nasi E
(1996b)
A slow current induced by light in rhabdomeric photoreceptors: possible relation to Icrac [abstract].
Biophys J
70:359.
-
Gomez M,
Nasi E
(1997)
Excitatory effects of activators of PKC on rhabdomeric photoreceptors[abstract].
Biophys J
72:92.
-
Hardie RC,
Minke B
(1992)
The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors.
Neuron
8:643-651[Web of Science][Medline].
-
Hardie RC,
Peretz A,
Suss-Toby E,
Rom-Glas A,
Bishop SA,
Selinger Z,
Minke B
(1993)
Protein kinase C is required for light adaptation in Drosophila photoreceptors.
Nature
363:634-637[Medline].
-
Heikkilä J,
Åkerman KEO
(1989)
()-Indolactam V activated protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells.
Biochem Biophys Res Commun
162:1207-1213[Web of Science][Medline].
-
Herbert JM,
Augereau JM,
Gleye J,
Maffrand JP
(1990)
Chelerythrine is a potent and specific inhibitor of protein kinase C.
Biochem Biophys Res Commun
172:993-999[Web of Science][Medline].
-
Hollander M,
Wolfe DA
(1973)
In: Nonparametric statistical methods. New York: Wiley.
-
Lisman JE,
Brown JE
(1975)
Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors.
J Gen Physiol
66:489-506[Abstract/Free Full Text].
-
McKay RR,
Chen D-M,
Miller K,
Kim S,
Stark WS,
Shortridge RD
(1995)
Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.
J Biol Chem
270:13271-13276[Abstract/Free Full Text].
-
Nasi E
(1991a)
Electrophysiological properties of isolated photoreceptors from the eye of Lima scabra.
J Gen Physiol
97:17-34[Abstract/Free Full Text].
-
Nasi E
(1991b)
Two light-dependent conductances in the membrane of Lima photoreceptor cells.
J Gen Physiol
97:55-72[Abstract/Free Full Text].
-
Nishizuka Y
(1984)
The role of protein kinase C in cell surface signal transduction and tumor promotion.
Nature
308:693-698[Medline].
-
Pak WL,
Grossfield J,
White NV
(1969)
Nonphototactic mutants in a study of vision in Drosophila.
Nature
222:351-354[Medline].
-
Payne R,
Fein A
(1986)
The initial response of Limulus ventral photoreceptors to bright flashes: released calcium as synergist to excitation.
J Gen Physiol
87:243-269[Abstract/Free Full Text].
-
Payne R,
Corson DW,
Fein A
(1986a)
Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors.
J Gen Physiol
88:107-126[Abstract/Free Full Text].
-
Payne R,
Corson DW,
Fein A,
Berridge MJ
(1986b)
Excitation and adaptation in Limulus ventral photoreceptors by inositol 1,4,5-trisphosphate result from a rise in intracellular calcium.
J Gen Physiol
88:127-142[Abstract/Free Full Text].
-
Payne R,
Walz B,
Levy S,
Fein A
(1988)
The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback.
Philo Trans R Soc Lond B Biol Sci
320:359-379[Abstract/Free Full Text].
-
Petersen CCH,
Berridge MJ
(1994)
The regulation of capacitative calcium entry by calcium and protein kinase C in Xenopus oocytes.
J Biol Chem
269:32246-32253[Abstract/Free Full Text].
-
Shin J,
Richard EA,
Lisman JE
(1993)
Ca2+ is an obligatory intermediate in the excitation cascade of Limulus photoreceptors.
Neuron
11:845-855[Web of Science][Medline].
-
Smith DP,
Ranganathan R,
Hardy RW,
Marx J,
Tsuchida T,
Zuker CS
(1991)
Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C.
Science
254:1478-1484[Abstract/Free Full Text].
-
Szuts EZ,
Wood S,
Reid MS,
Fein A
(1986)
Light stimulates the rapid formation of inositol trisphosphate in squid retinas.
Biochem J
240:929-932[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18145253-11$05.00/0
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Top
Abstract
Introduction
