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Volume 17, Number 5,
Issue of March 1, 1997
pp. 1701-1709
Copyright ©1997 Society for Neuroscience
Rapid Coupling of Calcium Release to Depolarization in
Limulus polyphemus Ventral Photoreceptors as Revealed by
Microphotolysis and Confocal Microscopy
Kyrill Ukhanov and
Richard Payne
Department of Zoology, University of Maryland, College Park,
Maryland 20742
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Microphotolysis and confocal microscopy were used to investigate
the timing of calcium release and of the electrical response in
Limulus polyphemus ventral photoreceptors. The
fluorescent dyes Fluo-3 and Calcium Green-5N were used to monitor local
Ca2+ elevations. Photolysis of caged inositol trisphosphate
(InsP3) close to the plasma membrane of the
light-sensitive rhabdomeral (R-) lobe resulted in Ca2+
elevation within 10-20 msec, 20-45 msec before the physiological response to light normally would be detected. Inward ionic current flow
and depolarization followed InsP3-induced calcium release within 2.5 ± 3.3 msec. Voltage-clamping the cells and removal of
extracellular Ca2+ did not affect the timing of the
Ca2+ elevation that followed the photolysis of caged
InsP3 or its relationship to the electrical response. In
contrast to the physiological response to light, which only released
calcium within the R-lobe, photolysis of InsP3 elevated
Cai in both lobes, although with much greater effect in the
R-lobe, as compared with the bulk of the A-lobe, suggesting the
presence of InsP3-sensitive calcium stores in both lobes.
Photolysis of caged calcium [o-nitrophenyl EGTA (NPE)]
at the edge of the R-lobe activated an inward ionic current within
1.8 ± 0.7 msec. This NPE-induced current reversed at a membrane
potential of 10 ± 6 mV in the range typical of that of the
light-activated current under physiological conditions. Calcium
release, therefore, activates an inward current rapidly enough to
contribute to the electrical response to light.
Key words:
phototransduction;
Limulus polyphemus;
photoreceptor;
nitrophenyl EGTA (NPE);
caged InsP3;
confocal microscopy
INTRODUCTION
The light response in invertebrate photoreceptors
is thought to be mediated by the ubiquitous phosphoinositide-signaling
(PI) pathway (Bloomquist et al., 1988 ; Hardie and Minke, 1995;
Ranganathan et al., 1995). The major known products of the PI pathway
are inositol 1,4,5 trisphosphate (InsP3), which releases
stored calcium ions, and diacylglycerol (Berridge, 1993). However,
there is no clear understanding of how the products of the PI pathway
can be linked to the opening of the ion channels that depolarize the membrane potential of the photoreceptor. For the ventral photoreceptors of the horseshoe crab (Limulus polyphemus), intracellular
pressure injections of InsP3 or Ca2+ ions
activate, in darkness, an inward current having a reversal potential
(Erev) similar to that of the light-activated
current (Brown et al., 1984; Fein et al., 1984). The injections also
subsequently desensitize the light response. Therefore, elevation of
the cytosolic Ca2+ ion concentration (Cai) is
thought to play a role in both excitation and adaptation of
Limulus photoreceptors (Frank and Fein, 1991; Nagy, 1991;
Shin et al., 1993; Contzen et al., 1995). However, it has not been
possible so far to verify that released calcium can activate ion
channels sufficiently rapidly to contribute to the generation of inward
current during the light response. In excised patches of
light-sensitive membrane, cyclic guanosine monophosphate (cGMP), but
not calcium ions, activates ion channels (Bacigalupo et al., 1991).
Calcium-activated production of cGMP has, therefore, been proposed to
couple the elevation of Cai to the activation of ion
channels (Shin et al., 1993). The proposal of this additional step
raises further doubts that calcium can act rapidly enough. Therefore,
the time taken for released calcium to activate an inward current is
clearly critical for determining the role of light-induced calcium
release in mediating the electrical response to light. To investigate
the timing of the response to released calcium ions, we have used
fluorescent calcium-sensitive dyes and photolysis of caged
InsP3 and nitrophenyl EGTA (NPE) to create and measure
spatially localized calcium transients with a millisecond time
resolution (Walker et al., 1989; Ellis-Davies and Kaplan,
1994).
MATERIALS AND METHODS
Ventral optic nerves were dissected as described by Millecchia
and Mauro (1969) and placed in artificial seawater (ASW) containing (in
mM): 435 NaCl, 10 KCl, 20 MgCl2, 25 MgSO4, 10 CaCl2, and 10 HEPES, pH 7.0. For some
experiments the preparation was kept on ice in ASW containing 40 mM hydroxylamine for 15 min under bright white light
(Faddis and Brown, 1992). After treatment with 1% Pronase, ventral
photoreceptor cells were impaled with a glass micropipette. A solution
containing 10-25 mM GDP- S
[guanosine-5 -O-(2-thiodiphosphate); Calbiochem, La Jolla,
CA], 1 mM Fluo-3 or Calcium Green-5N (Molecular Probes,
Eugene, OR), 10 mM caged InsP3 (Calbiochem),
100 mM K-aspartate, and 10 mM HEPES, pH 7.0, was pressure-injected from the micropipette into the cells.
Approximately 50-100 injections of 1-10 pl were delivered before the
beginning of the experiment. Caged ATP (10 mM; Calbiochem)
replaced caged InsP3 in the injection solution for control
experiments.
For experiments using caged calcium, 40 mM NPE
(o-nitrophenyl EGTA hexapotassium salt; Molecular Probes)
mixed with 32 mM CaCl2 replaced caged
InsP3 in the micropipette solution. A 0.8 Ca-NPE mixture
should yield a Ca2+ concentration of ~1 µM
(Ellis-Davies and Kaplan, 1994). After multiple UV flashes,
Cai was increased irreversibly in some cells, presumably
because of the cumulative shift in the Ca:NPE ratio. This sustained
elevation of Cai was associated in some cells with a
sustained inward current (Shin et al., 1993).
Ventral nerves were viewed with a Zeiss LSM 410 laser-scanning confocal
microscope equipped with a 488 nm argon laser (Uniphase) focused
through a Zeiss Neofluor 40×/0.75 objective lens (for details, see
Ukhanov and Payne, 1995). A 351/364 nm ion argon laser (Innova
Technologies) also was focused through the same objective lens and was
used to photolyze caged compounds. The UV laser intensity was
attenuated by neutral density filters and is expressed here by an
arbitrary relative scale. High-speed shutters (Uniblitz model 26L,
Vincent Associates, Rochester, NY) were placed in the path of the laser
beams to control the timing and duration of flashes. A procedure
similar to that described by Wang and Augustine (1996) was used to
check that the focus and position of the UV spot were coaxial with the
488 nm laser beam. Briefly, photobleaching of a thin polysterene film
stained with Nile Red was used to determine the position of the focused
spots created by the 488 nm and UV lasers.
For line scans, the laser beam swept every 4 msec across the cell, each
scan containing 512 pixels. In all, 512 successive scans were stacked
to create a raw image of fluorescence that represented 2.048 sec of
recording time. Changes of Cai on illumination of the
photoreceptors were displayed as a ratio of fluorescence relative to
the fluorescence, Fo, recorded during the latent
period of the response. National Institutes of Health IMAGE software (written by Wayne Rasband at National Institutes of Health and available via anonymous ftp from zippy.nimh.nih.gov) was used for
off-line image processing, including smoothing and ratio calculations. Smoothing reduced the spatial resolution of line scans to 4 µm. By
photobleaching Nile Red-stained polystyrene film, we also determined the uniformity of illumination during laser beam scans. We found that
the intensity of the laser beams varied by <30% over the first 412 pixels of the scan. However, the beam intensity increased fourfold over
the final 100 pixels because of a slowing of the movement of the beam
toward the end of each scan. To avoid artifacts because of increased
photolysis in this region of the scan, we deleted from our analysis the
100 pixels at the right-hand side of every line in the stack.
Membrane voltage or ionic current were recorded with an
Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) and digitally sampled at a rate of at least 1 kHz. For current recording and measuring of the Erev under two-electrode
voltage clamp, the cells were impaled with a second micropipette filled
with 3 M KCl (resistance < 10 M ).
RESULTS
Use of GDP-S and hydroxylamine to prolong response latency
We previously have used steps of light delivered by the 488 nm
laser of our confocal microscope to excite ventral photoreceptors and
simultaneously to monitor Cai (Ukhanov and Payne, 1995). To release caged compounds, we delivered a flash from a 364 nm laser at
the onset of the 488 nm step, both lasers being focused onto the same
point (see Materials and Methods). The application of caged compounds
to functioning photoreceptor cells presents a challenge, because
rhodopsin absorbs the UV light required for photolysis. This would be
expected to accelerate calcium release and the electrical response
through the normal physiological pathway. It is, therefore, important
to reduce the sensitivity of the physiological pathway as much as
possible. In the case of Limulus ventral photoreceptors, the
physiological response to the intense laser light delivered by our
confocal microscope is delayed by a latent period of ~20 msec
(Ukhanov and Payne, 1995). To reduce the gain of phototransduction and
to increase the latent period even further, we followed the protocol of
Faddis and Brown (1992), treating the cells with hydroxylamine and
injecting them with GDP-S. By bleaching rhodopsin (Hubbard and Wald,
1960) and inhibiting GTP-binding proteins (Fein, 1986), respectively,
these agents slow excitation of the cells by light without affecting
the response to injected InsP3 (Faddis and Brown, 1992). As
a result, the latency of the normal physiological response to both the
488 nm laser and the flash from the UV laser is prolonged to 30-50
msec (Payne and Ukhanov, 1996). This latency enabled us to observe the
effects of the release of caged compounds, which photolyze within 1-3
msec, before any physiological response caused by the activation of
rhodopsin is detectable.
Photolysis of caged InsP3 rapidly elevates
Cai in ventral photoreceptors
Ventral photoreceptors were injected with GDP-S, caged
InsP3, and the calcium indicator dye Fluo-3 and were viewed
with a laser- scanning confocal microscope. After orientation of the laser beam, a line scan was performed in a plane that passed through the interior of the light-sensitive rhabdomeral (R-) lobe of the photoreceptor, the insensitive arhabdomeral (A-) lobe (Calman and
Chamberlain, 1982), and the axon (Fig.
1A). The 488 nm laser used for this
line scan excited the cell through the normal physiological mechanism
without photolyzing the cage and simultaneously elicited fluorescence
from Fluo-3 so as to monitor accompanying changes in Cai.
After a latent period of 40 msec, the elevation of Cai began at the extreme edge of the R-lobe beneath the photoreceptive microvillar membrane (Ukhanov and Payne, 1995) and then spread over the
next 400 msec toward the boundary between the R-lobe and the
light-insensitive A-lobe (Fig. 1B, upper
frame). The elevation of Cai barely penetrated the
A-lobe. In five cells, Fluo-3 fluorescence recorded during illumination
by the 488 nm laser within 10 µm of the edge of the R-lobe rose to a
peak of 3.7 ± 0.8 times its initial level (mean ± SD). At
70 µm from the edge of the R-lobe, the fluorescence remained at
1.1 ± 0.11 times its initial level over the same time period. A
10 msec flash from a UV laser then was superimposed at the beginning of
a second 488 nm scan. This flash initiated fast cleavage of caged
InsP3, with consequent elevation of Cai within
30 msec along the entire length of the scanned line within the R-lobe
(Fig. 1B, lower frame). The elevation of
Cai along the scanned line in the A-lobe was much less than that in the R-lobe, with the exception of a region close to the axon.
For the same five cells, the peak Fluo-3 fluorescence after the UV
flash recorded within 10 µm of the edge of the R-lobe was 4.0 ± 0.7 times its initial level (mean ± SD). At 70 µm from the edge
of the R-lobe, the peak fluorescence was 1.6 ± 0.6 times its
initial level.
Fig. 1.
Photolysis of caged InsP3 initiates
calcium release throughout Limulus ventral
photoreceptors. A, Ventral photoreceptor loaded with 10 mM GDP-S, 1 mM Fluo-3, and 10 mM
caged InsP3. The laser beam scanned along the green
line every 4 msec, and the resulting lines of fluorescence data
were stacked to create the images below. B, Upper frame, Fluorescence recorded
during a line scan with a 488 nm laser beam, which excited the
photoreceptor through the physiological mechanism. The scan shows the
temporal progression of the physiological elevation of Cai
from the edge of the R-lobe membrane (left-hand edge of
scan) into the A-lobe (right-hand side of scan). The
laser beam started scanning across the cell at a time indicated by the
first line of the image. The left-hand side of each
scanned line was cropped to eliminate pixels beyond the edge of the
R-lobe (determined as the point at which total fluorescence drops by
50%). The right-hand edge of each scanned line also was
cropped to eliminate the final 100 pixels in which laser illumination
was not uniform (see Materials and Methods). B,
Lower frame, A 10 msec flash from a UV (351/364 nm)
laser, relative intensity 0.5 (red line), was
superimposed at the beginning of the scan. The resulting photolysis of
caged InsP3 induced fast calcium release throughout the
cell. C, To exclude any contribution of Ca2+
influx, we kept a different cell in darkness for 30 min in ASW containing 1 mM EGTA instead of 10 mM
Ca2+. This increased the latency of response to the 488 nm
laser via the physiological mechanism (upper
frame) but did not alter the latency or pattern of elevation of
Cai after photolysis of InsP3 (lower
frame).
[View Larger Version of this Image (41K GIF file)]
To verify that most of the Cai rise comes from the release
of internal calcium stores, we removed Ca2+ from the ASW
bathing another ventral nerve. The nerve was kept in darkness for 30 min in ASW containing 1 mM EGTA instead of 10 mM Ca2+. As expected from previous work
(Lisman, 1976; Payne and Flores, 1992), removal of extracellular
Ca2+ increased the latent period of the physiological
response to the 488 nm scan (Fig. 1C, upper
frame) to 230 msec. Nevertheless, a brief UV flash elicited as
rapid a response to photolysis of InsP3 along the entire
scanned line as for cells bathed in 10 mM Ca2+
(Fig. 1C, lower frame). These results might be
explained if prolonged exposure of the cell to zero extracellular
Ca2+ and the accompanying reduction of resting levels of
Cai (Levy and Fein, 1985; Ukhanov et al., 1995) resulted in
a slowing of light-induced phospholipase-C activity (Rack et al., 1994;
Mitchell et al., 1995).
Rapid coupling of InsP3-induced elevation of
Cai to the electrical response
Positioning the laser beam at a stationary spot allowed detection
of fluorescence with the highest temporal resolution as well as
microphotolysis at the fastest rate. The spot chosen was located at the
edge of the R-lobe where the fastest elevations of calcium
via the physiological mechanism were observed. Because this
placement was based on the spatially smoothed line scan images of
calcium release, resolution of the edge of R-lobe was limited to ~4
µm (see Materials and Methods). During a 488 nm flash delivered to
this spot, calcium release via the physiological mechanism began after
a latent period of 42 msec and rapidly saturated the Fluo-3 (Fig.
2A). Membrane depolarization was
detected simultaneously with the calcium release. A brief superimposed
UV flash, which photolyzed caged InsP3, initiated calcium
release with a greatly reduced latency of 13 msec (Fig.
2B). Membrane depolarization followed 2 msec later.
We attribute this earlier calcium elevation and electrical response to
photolytic release of InsP3 by the UV flash.
Fig. 2.
Timing of calcium release by using the stationary
spot mode of the laser. Photoreceptors were loaded with either Fluo-3
or Calcium Green-5N, caged InsP3, and GDP-S. The 488 nm
and UV (351/364 nm) laser beams were focused onto the edge of the
R-lobe, equivalent to the extreme left-hand edge of the
photoreceptor in Figure 1A. A,
Membrane potential (solid line) and Fluo-3 fluorescence
(dots) recorded during illumination by the 488 nm laser.
Laser stimulation started at the beginning of the fluorescence trace.
B, Effect of superimposing a 20 msec duration UV flash.
C, Membrane potential and Calcium Green-5N fluorescence
recorded from a photoreceptor illuminated by the 488 nm laser.
D, Effect of superimposing a 3-msec-duration UV flash.
The rapid reduction in fluorescence in B and
D on termination of the UV flash is attributable to
cessation of autofluorescence and additional Fluo-3 fluorescence
created by the UV illumination; relative intensity 0.05 in
B and 0.5 in D. Fluorescence was sampled
every 0.9 msec in A and C and every 0.2 msec in B and D.
[View Larger Version of this Image (20K GIF file)]
The low-affinity calcium indicator dye Calcium Green-5N (Haugland,
1992) was used to better compare peak elevations of Cai. Calcium Green-5N saturates at much higher levels of Cai
than Fluo-3 and is therefore more suitable for following the time
course and magnitude of large elevations of Cai. The 488 nm
laser again was focused onto a spot at the edge of the R-lobe. Calcium
Green-5N fluorescence during a 488 nm step rose similarly to the Fluo-3 signals (Fig. 2C), reaching a peak within ~100 msec.
Photolysis of caged InsP3 at the same spot induced a peak
fluorescence increase that was nearly threefold larger than that
activated by 488 nm light alone (Fig. 2D) and reached
its peak within 20 msec, implying an extremely fast and large
Cai rise.
We investigated the effect of removing extracellular Ca2+
and of voltage-clamping photoreceptors. Figure 3 shows
inward current and fluorescence recorded from a voltage-clamped
photoreceptor that had been filled with Fluo-3, GDP-S, and caged
InsP3. Before stimulating the cell, the photoreceptor was
bathed in darkness for 30 min in ASW containing 1 mM EGTA
instead of 10 mM CaCl2. As expected from the
line scans (Fig. 1C), the latent period of the response to
the 488 nm flash alone (Fig. 3A), but not to the superimposed UV flash (Fig. 3B), was greatly prolonged, as
compared with control conditions (compare Figs. 2 and 3, noting the
different time scales). The relationship between the timing of the
electrical response and the fluorescence traces that followed
photolysis of caged InsP3 by the UV flash was also similar
to that seen under control conditions (Fig. 3, inset).
Removal of extracellular calcium for many minutes therefore did not
greatly affect the coupling of InsP3-induced calcium
release to the generation of inward current.
Fig. 3.
Removal of extracellular Ca2+ does not
affect InsP3-induced calcium release in
Limulus ventral photoreceptors. A,
Membrane current (solid line) and Fluo-3 fluorescence
(dots) recorded from a photoreceptor voltage-clamped to
its resting membrane potential and illuminated by a 488 nm laser. The
photoreceptor was bathed in artificial seawater containing 1 mM EGTA instead of 10 mM Ca2+. Note
the change in time scale, as compared with Figure 2. B, Effect of superimposing a 40 msec duration UV flash. The rapid reduction in fluorescence in B on termination of the UV
flash is attributable to cessation of autofluorescence and additional Fluo-3 fluorescence created by the UV illumination; relative intensity 0.5. Fluorescence was sampled every 0.9 msec.
[View Larger Version of this Image (13K GIF file)]
For further analysis of the data obtained in normal ASW, we compared
the latency of the electrical response and of the Cai elevation induced by the photolysis of caged InsP3 at the
edge of the R-lobe. The complete data are plotted as a scatter diagram in Figure 4 (solid circles). The mean
latency of the calcium signals (the first detectable increase in Fluo-3
fluorescence) that followed photolysis of caged InsP3 by
the UV flash was 17 ± 6 msec (mean ± SD; n = 42; 14 cells), whereas the mean latency of the electrical response
was 20 ± 6 msec. The variable latency of the
InsP3-induced calcium signal may be a general property of
InsP3-induced calcium release, shared with other cells
(Parker and Ivorra, 1993). However, the relative timing of the calcium
signal and the electrical response exhibited less variability. On
average, the InsP3-induced calcium signal led the
electrical response by 2.5 ± 3.3 msec.
Fig. 4.
A scatter plot of the latencies of the calcium
signal (both Fluo-3 and Calcium Green-5N fluorescence) and of the
electrical responses (either photocurrent or changes in membrane
potential) after photolysis of caged InsP3
(filled circles) or during the physiological
response to 488 nm light (open circles). A
straight diagonal line indicating equality of the two
latencies is drawn to show the slightly different distributions of the
two sets of data points.
[View Larger Version of this Image (22K GIF file)]
Latencies for the responses to physiological stimulation of the
cell by 488 nm illumination alone were longer than those for the
responses to the release of caged InsP3 (Fig. 4, open
circles). The mean latency of the physiological electrical
response was 45 ± 12 msec (n = 52; 14 cells),
whereas the latency of the accompanying calcium signals was 48 ± 12 msec. The comparatively large SD of these data reflect, in part, the
known variability inherent in the process that determines the latency
of the physiological response to individual quanta (Yeandle and
Spiegler, 1973) including, presumably, the time taken to generate
InsP3 and for InsP3 to release calcium (see
above). The distribution of the latencies also differed from those
elicited after photolysis of caged InsP3. The majority (32 of 52) of the latency data for the physiological responses falls above
the diagonal line in Figure 4, indicating that the detection of calcium
release often slightly lagged the initiation of the physiological
electrical response. One explanation for the difference in the
distributions of latencies might be the time taken for calcium to
diffuse small distances. Even at the highest diffusion rate of 227 µm2/sec (Allbritton et al., 1992), Ca2+ ions
will take ~3 msec to travel 1 µm. The resolution of the edge of the
R-lobe, determined from line scan images of calcium release, was
limited by spatial smoothing to ~4 µm. Thus, the confocal spot
could have been placed a few micrometers from the microvillar membrane
of the R-lobe. This error in placement might account for the systematic
difference in the timing of the physiological and
InsP3-induced calcium signals. Calcium released by the
physiological mechanism would be expected to be initiated from calcium
stores directly beneath the microvillar membrane, where light-induced production of InsP3 occurs. This physiological release of
calcium immediately would open channels in the plasma membrane.
However, if the confocal measuring spot were placed more than a
micrometer from the microvillar membrane, the calcium ions would have
to diffuse to the spot to be detected. The detection of the calcium signal would, therefore, be expected to lag the physiological electrical response by a few milliseconds. On the other hand, calcium
ions released by photolysis of caged InsP3 from stores at
the same distant confocal spot would be detected immediately, but the
calcium ions would have to diffuse to the microvillar membrane to
initiate an electrical response. If the confocal measuring spot were
placed more than a micrometer from the microvillar membrane, the
calcium signal might, therefore, be expected to lead the electrical response to photolysis of caged InsP3 by a few milliseconds
but to lag the physiological electrical response to 488 nm light.
Photolysis of caged ATP had little effect on calcium release or the
electrical response
We were concerned that the absorption of UV light by rhodopsin, in
addition to the release of caged InsP3, might accelerate the response of the cell when the UV flash was superimposed on the 488 nm step. We therefore substituted caged ATP for caged InsP3
in the solution injected into the cells and repeated some of the above
experiments. Responses from cells coinjected with 10 mM
caged ATP, Fluo-3, and GDP-S did not exhibit a large reduction in
the latency of their response when the UV flash was superimposed on the
488 nm flash (Fig. 5). The mean latencies of the calcium signals after the 488 nm laser flash were 42 ± 2 msec without the
UV flash and 39 ± 5 msec (n = 8) with the
superimposed UV flash (relative intensity 0.5, duration 30 msec),
whereas those of the electrical responses were 37 ± 5 and 33 ± 7 msec, respectively. We presume that the UV flash is relatively
ineffective because the 488 nm illumination alone locally saturates the
physiological response to light. The UV flash, however, does elicit
increased autofluorescence and Fluo-3 fluorescence (Fig.
5B). Because the UV flash, by itself, does not significantly
alter the initial response of the cell to the 488 nm light, we
attribute the effects of UV stimulation of cells loaded with caged
compounds to the release of active InsP3 or free
Ca2+.
Fig. 5.
Photolysis of caged ATP does not alter either
electrical excitation or light-induced calcium release in
Limulus ventral photoreceptors. Photoreceptors were
loaded with Fluo-3, caged ATP, and GDP-S. The 488 nm and UV (351/364
nm) laser beams were focused onto the edge of the R-lobe, equivalent to
the extreme left-hand edge of the photoreceptor in
Figure 1A. A, Membrane potential
(solid line) and Fluo-3 fluorescence
(dots) recorded during illumination by a 488 nm laser.
Laser stimulation began at the beginning of the fluorescence trace.
B, Effect of superimposing a 30 msec UV flash; relative
intensity 0.5. The break in the vertical scale
represents 7.5 F/Fo units and
is needed to show the time course of autofluorescence excited by the UV
light.
[View Larger Version of this Image (12K GIF file)]
Use of caged calcium
To determine the speed of action of released Ca independently, we
directly released calcium from NPE. Injection of photoreceptors with 40 mM 0.8 Ca-NPE decreased the latent period of the
physiological response to 488 nm light to <30 msec (compare Fig.
6B). This effect can be ascribed to
the higher Cai, 1 µM, in the injection
solution, as compared with the normal resting Cai of 0.4 µM, because calcium elevation is known to reduce response
latency (Fein and Charlton, 1977). NPE injection also slowed the rise
time of the fluorescence increase caused by calcium released
via the physiological mechanism [compare the fluorescence
trace in Fig. 2A with the control (UV = 0) trace in Fig. 6A]. We ascribe this to the
buffering of Cai by NPE. Photolytic release of
Ca2+ ions in the vicinity of the photoreceptor membrane by
a 3 msec UV flash induced an inward current that clearly consisted of
more than one component (traces labeled UV = 1.0, UV = 2.5 in Fig. 6B). An early transient inward
current, rising to peak within 10 msec, was observed only after the UV
flash and was attributed to the photolysis of NPE. The physiological
response to the 488 nm light would not be expected to exhibit such an
early transient current because of the 30-60 msec latent period
associated with the physiological release of calcium (traces labeled
UV = 0 in Fig. 6). A later larger current component
induced by the UV flash rose at approximately the same time as the
response to the 488 nm step and was, therefore, attributed to the
physiological response. To verify that most of this early ionic current
flows via ion channels rather than via the activation of an
electrogenic Na/Ca exchanger, we determined its reversal potential
under voltage clamp. Reversal of the early NPE-induced current is shown
in Figure 6C. The reversal potential
(Erev) lay between 3 and 22 mV, averaging 10 ± 6 mV (n = 4). The bulk of the current that
followed the early NPE-induced transient reversed at a slightly more
positive potential in all cells, averaging 19 ± 6 mV. In separate
experiments, reversal of the inward current activated by the photolysis
of caged InsP3 also was observed (data not shown). At high
intensities of the UV laser, the latency of the electrical response to
photolysis of caged NPE followed the onset of the UV flash by 1.8 ± 0.7 msec (mean ± SD, 5 cells). There is, therefore, a good
agreement with the 2.5 ± 3.3 msec delay of the electrical
response with respect to the calcium signal after photolysis of caged
InsP3.
Fig. 6.
UV flashes activated an early inward ionic current
in Limulus ventral photoreceptors loaded with 40 mM 0.8 Ca-NPE, Fluo-3, and GDP-S. A 3 msec flash from a
UV (351/364 nm) laser was superimposed on a 488 nm laser step. The
relative intensity of the UV laser is as indicated in A.
Both lasers were focused on a spot at the edge of the R-lobe of a
ventral photoreceptor. A, Fluo-3 fluorescence, plotted
as the ratio of the Fluo-3 fluorescence, F, observed
during the response to that, Fini, sampled
before the onset of the first UV flash in the series delivered to the
cell. The detection of the elevation of Cai induced
via the normal physiological mechanism by the 488 nm
laser alone (UV = 0) was slowed significantly as a
result of the loading of the photoreceptor with NPE. The control trace
(UV = 0) was recorded after the presentation of the
UV flashes. B, Inward current activated within the 3 msec duration of the UV flashes. C, Reversal of inward
ionic current generated in another cell loaded with NPE and GDP-S
and illuminated by a 3 msec UV laser flash; relative intensity 50. For
this cell, Erev of the early current
transient was estimated as +22 mV.
[View Larger Version of this Image (27K GIF file)]
DISCUSSION
Faddis and Brown (1992) investigated the electrical response of
ventral photoreceptors after flash photolysis of InsP3
produced by diffuse illumination. They observed that release of caged
InsP3 increased the peak current produced through the
physiological mechanism via the activation of rhodopsin.
However, they could not separate the InsP3-induced current
from the physiological light-induced current. Our confocal method
reduces overall illumination of the photoreceptor while concentrating
the UV light. This technique reduces the overall stimulation of the
cell via the physiological mechanism while maximizing the
concentration of InsP3 released at its site of action. As a
result, we were able to observe an electrical response to the release
of caged InsP3 during the latent period when no
physiological response to light is detectable. Our results confirm
their main conclusion that the 10-20 msec delay between photolysis of
InsP3 and the electrical response indicates that the latter
is not caused by the direct rapid interaction of InsP3 with
a channel in the microvillar membrane. Rather, our simultaneous
measurements of Cai indicate that the electrical response
immediately follows and is caused by the release of Ca2+
ions (Payne et al., 1986).
Location of InsP3-sensitive calcium stores
Photolysis of caged InsP3 released calcium from stores
within both the lobes of Limulus ventral photoreceptors. We
note that not all of the calcium release necessarily passes through
InsP3-gated channels in the endoplasmic reticulum. Some
might be the consequence of a secondary wave of calcium-induced calcium
release. The consequent elevation of Cai is larger in the
R-lobe than in most regions of the A-lobe, with the exception of an
area close to the axon. Earlier experiments using aequorin as a calcium
indicator failed to reveal any significant calcium release on
microinjection of InsP3 into the A-lobe (Payne and Fein,
1986). More sensitive calcium-selective microelectrode recordings,
however, detected a relatively slow and small (~2 µM)
Ca2+ transient after injection of 100 µM
InsP3 into the A-lobe (Levy and Payne, 1993). Our results
support the latter observations and suggest that the aequorin imaging
system was not sufficiently sensitive to detect release in the A-lobe.
Our results are also consistent with a recent anatomical study showing
that smooth endoplasmic reticulum (SER) is not confined to the
submicrovillar region but that it forms a dense continuous network that
extends from beneath the microvillar membrane into the center of the
R-lobe. A less dense tubular reticulum, continuous with SER in the
R-lobe, extends into the A-lobe (Feng et al., 1994). The 488 nm light would result in InsP3 production at the microvillar plasma
membrane of the R-lobe through the PI pathway, creating a wave of
calcium release from the SER that spreads into the center of the R-lobe and diminishes in magnitude as the InsP3 diffuses into the
A-lobe. Release of caged InsP3, however, would elevate
Cai throughout the cell, with the magnitude of the
elevation of Cai in different cell regions being graded
according to the density of the SER. This might explain the larger
elevation of Cai in the R-lobe, as compared with the bulk
of the A-lobe.
The prominent InsP3-induced calcium release in the region
of the A-lobe where the axon originates suggests another concentration of SER there. We know of no published study on the distribution of SER
in this particular region of Limulus photoreceptors.
However, the presence of prominent ER and of the InsP3
receptor protein in the axons of mammalian neurons has been well
documented (Broadwell and Cataldo, 1984; Mignery et al., 1989). Because
we do not detect any elevation of calcium concentration in the axon
during the first second of the physiological response to light (Fig.
1B), we assume that the calcium stores in the axon
are not involved in the immediate transmission of the light response
along the axon. However, it may be that, during more prolonged
illumination, InsP3 produced in the cell body may diffuse
down the axon and regulate axonal function or structure. Alternatively,
the calcium stores in the axon may be vesicles that are being
transported to fulfill a role in the function of the axon terminal.
Speed of coupling of calcium release to depolarization
If, as is indicated by experiments on excised patches (Bacigalupo
et al., 1991), calcium does not bind directly to and open ion channels
in the microvillar membrane, then the intermediate steps that couple
calcium to the activation of ion channels impose a delay of only 1-3
msec. The significance of this result for visual excitation depends on
when calcium is released during the light response. Analysis of the
control responses to 488 nm illumination (Fig. 4, open
circles) indicates that the mean latency of the physiological
electrical response was 45 ± 12 msec, whereas the elevation of
Cai was first detected after 48 ± 12 msec. Because it
then takes ~50 msec for the photocurrent to reach its peak, there
seems to be sufficient time for released calcium ions to contribute to
the activation of the photocurrent during the rising edge of the
response to light. This assertion is consistent with the large
attenuation of the rate of rise of the photocurrent produced by the
intracellular injection of calcium chelators such as EGTA (Payne and
Fein, 1986) and di-Bromo-BAPTA (Shin et al., 1993). However, because
the detection of the physiological calcium signals often lagged that of
the electrical response by a few milliseconds, we cannot assert that
calcium elevation is the sole initiator of the electrical response. As
noted in Results, a simple explanation for this lag is the time taken
for the diffusion of calcium into the confocal spot after its release
from SER immediately below the microvillar membrane. However, it is
also possible that a parallel pathway of visual excitation exists, one
which can initiate an electrical response but which does not require
calcium release (Frank and Fein, 1991; Contzen et al., 1995). The
possibility of such a pathway is discussed further below.
Reversal potential of the calcium-activated current
Previous experiments in which calcium was pressure-injected into
the R-lobe concluded that both the calcium-induced current and the
light-induced current reversed between +10 and +20 mV (Payne et al.,
1986). The present experiments confirm this approximate range for the
reversal potentials but indicate a consistent difference between the
reversal potentials of current components activated by photolysis of
caged calcium and by the physiological mechanism in the presence of
NPE. The early transient, ascribed to calcium release, reversed at
10 ± 6 mV, whereas the much larger later component, ascribed to
the physiological mechanism, reversed at 19 ± 6 mV. This
difference in Erev may be significant. The
response of dark-adapted ventral photoreceptors to bright light flashes does not reverse at a unique potential and has been interpreted as
consisting of three components, each having a
Erev differing by a few millivolts (Deckert et
al., 1992). The second of these components, C2, reverses at
a potential a few millivolts below that of the other components.
Pharmacological experiments (Nagy, 1991; Contzen and Nagy, 1995) have
suggested that the C2 is mediated via
InsP3-induced calcium release, whereas the other components possibly are mediated via cyclic nucleotide metabolism. If
multiple pathways of excitation exist, then under the conditions of our flash photolysis experiment we would expect the buffering of calcium by
NPE to reduce the contribution of calcium release to the physiological response relative to other mechanisms. According to the proposal of
Nagy and collaborators, the early calcium-activated component of the
response to the photolysis flash may, therefore, be expected to display
a more negative reversal potential than the later physiological response.
Nagy and collaborators ascribe the different components of the light
response to the activation of different channels by the various
messenger pathways (Deckert et al., 1992). We think it is unlikely that
further analysis of small differences in reversal potential will yield
a definitive answer as to whether more than one channel mediates the
light response. A very large increase in Cai accompanies
the light response (Ukhanov and Payne, 1995), and the light-activated
channels may be calcium-permeable, or the voltage-gated channels that
mediate the current induced by depolarizing voltage steps may be
calcium-sensitive. These factors may distort the waveform of the
photocurrent close to the reversal potential (O'Day et al., 1993). A
future combination of local flash photolysis of NPE with patch-clamp
recording, however, may allow the direct comparison of single channels
activated by released calcium and by the physiological mechanism under
different conditions.
Comparison with Drosophila photoreceptors
It is noteworthy that experiments recently performed on
Drosophila photoreceptors with similar methods have failed
to observe any ionic current directly activated by the photolysis of
caged calcium. Flash photolysis of DM-Nitrophen loaded by dialysis into dissociated Drosophila photoreceptors activates only the
Na/Ca exchanger (Hardie, 1996). Instead, a physical link has been
proposed between the activation of the InsP3 receptor and
the opening of the putative channel protein TRP (Hardie and Minke,
1995). This difference may indicate that microvillar photoreceptors of
invertebrates have evolved a variety of linkages of the PI pathway to
ion channel activation.
FOOTNOTES
Received Sept. 3, 1996; revised Dec. 20, 1996; accepted Dec. 23, 1996.
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, for the use of the confocal
microscope and Drs. Roger Hardie and Mark Gray-Keller for their
comments.
Correspondence should be addressed to Dr. Richard Payne, Department of
Zoology, University of Maryland, College Park, MD 20742.
Dr. Ukhanov's present address: Institut fuer Zellphysiologie,
Universitaet Potsdam, Lennestrasse 7a, D-14471 Potsdam,
Germany.
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