 |
 Previous Article | Next Article 
The Journal of Neuroscience, April 1, 1998, 18(7):2467-2474
Calcium Extrusion from Mammalian Photoreceptor Terminals
Catherine W.
Morgans1, 2,
Oussama
El Far2,
Amy
Berntson1,
Heinz
Wässle1, and
W. Rowland
Taylor1
Departments of 1 Neuroanatomy and
2 Neurochemistry, Max-Planck-Institute für
Hirnforschung, D-60528 Frankfurt, Germany
 |
ABSTRACT |
Ribbon synapses of vertebrate photoreceptors constantly release
glutamate in darkness. Transmitter release is maintained by a steady
influx of calcium through voltage-dependent calcium channels, implying
the presence of a mechanism that is able to extrude calcium at an equal
rate. The two predominant mechanisms of intracellular calcium extrusion
are the plasma membrane calcium ATPase (PMCA) and the
Na+/Ca2+-exchanger.
Immunohistochemical staining of retina sections revealed strong
immunoreactivity for the PMCA in rod and cone terminals, whereas
staining for the
Na+/Ca2+-exchanger was very weak.
The PMCA was localized to the plasma membrane along the sides of the
photoreceptor terminals and was excluded from the base of the terminals
where the active zones are located. The amplitude of a
calcium-activated chloride current was used to monitor the
intracellular calcium concentration. An upper limit for the time
required to remove intracellular free calcium is obtained from two time
constants measured for the calcium-activated chloride current tail
currents: one of 50 msec and a second of 190 msec. Calcium extrusion
was inhibited in the absence of intracellular ATP or in the presence of
the PMCA inhibitor orthovanadate, but was unaffected by replacement of
external Na+ with Li+. The data
indicate that the PMCA, rather than the
Na+/Ca2+-exchanger, is the
predominant mechanism for calcium extrusion from photoreceptor synaptic
terminals.
Key words:
retina; photoreceptors; calcium extrusion; Ca2+-ATPase; Na+/Ca2+ exchanger
| |
INTRODUCTION |
Neurotransmitter release within the
CNS is triggered by pulses of calcium through voltage-gated calcium
channels that open in response to depolarization of the plasma
membrane. On closure of the channels, the calcium concentration drops
and transmitter release declines. Free calcium at the active zone
decreases rapidly through diffusion, sequestration by intracellular
calcium binding proteins, and ultimately extrusion across the plasma
membrane. The rise in calcium in most CNS synaptic terminals is brief
because of the short duration of action potential-induced
depolarizations (Katz and Miledi, 1967 ).
In contrast, ribbon synapses, which occur in photoreceptor and bipolar
neurons of the retina and in hair cells of the inner ear, are
characterized by tonic influx of calcium in response to sustained
depolarization (DeVries and Baylor, 1993). Photoreceptors, for example,
are constantly depolarized in darkness, their presynaptic calcium
channels remain open, and glutamate is continually released (Copenhagen
and Jahr, 1989). They respond to light by hyperpolarizing and
decreasing the rate of transmitter release. Voltage-dependent calcium
channels transduce the hyperpolarization into a decrease in the influx
of calcium ions, resulting in a decrease in the intracellular calcium
concentration.
The constant influx of calcium into photoreceptors during darkness
implies the existence of an efficient calcium extrusion mechanism that
exactly balances the rate of calcium entry. This prevents calcium
levels from rising in the soma and ensures a rapid decrease in the
calcium concentration in the terminal in response to light. By
measuring membrane turnover in response to the release of caged
intracellular calcium, Lagnado and coworkers (1996) and Rieke and
Schwartz (1996) found that raising intracellular calcium to <5
µM was sufficient to trigger synaptic vesicle exocytosis from retinal ribbon synapses. These studies suggest that transmitter release might be governed by the average cytosolic calcium
concentration rather than calcium microdomains in the vicinity of
active channels. If this is true, then the calcium extrusion mechanism
could well be an important determinant of the response kinetics of
photoreceptor synapses.
In neurons, two mechanisms with somewhat different properties are
responsible for extruding calcium ions and returning the calcium
concentration to baseline levels (DiPolo and Beauge, 1979; Blaustein,
1988). One is a low-affinity
Na+/Ca2+ exchanger that is linked
to the sodium electrochemical gradient. For each calcium pumped out,
three sodium ions enter the cell (Reeves and Hale, 1984). The
concentration gradients supporting this system are maintained by the
Na+/K+-ATPase. A specialized form
of the Na+/Ca2+-exchanger, which
co-transports potassium ions, is responsible for extruding calcium ions
from the outer segments of photoreceptors (Lagnado et al., 1988). The
other extrusion mechanism is a high-affinity plasma membrane
Ca2+-ATPase (PMCA) that can generate large
concentration gradients, limited by the free energy change of ATP
hydrolysis. One ATP molecule is hydrolyzed for every calcium ion pumped
out (Niggli et al., 1981).
The aim of the present study was to determine which of these two
mechanisms is responsible for removing calcium from the synaptic terminals of retinal photoreceptors.
| |
MATERIALS AND METHODS |
Antibodies. The mouse monoclonal antibody against the
plasma membrane Ca2+-ATPase was purchased from Sigma
(Deisenhofen, Germany). The rabbit polyclonal antiserum against the
Na+/Ca2+ exchanger was from Swiss
Antibodies. The rabbit polyclonal antibody against synaptobrevin was
kindly provided by Dr. M. Takahashi (Mitsubishi Kasei Institute of Life
Sciences). The following secondary antibodies were used: goat
anti-mouse IgG conjugated to CY3 (carboxymethylindocyanine; Dianova,
Hamburg, Germany), goat anti-rabbit IgG conjugated to FITC (fluorescein
isothiocyanate; Dianova), goat anti-mouse IgG conjugated to FITC
(Dianova), and donkey anti-sheep IgG conjugated to CY3 (Dianova). The
syntaxin-3 antibody is described elsewhere (Morgans et al., 1996). The
calcium channel antibody D1a was raised against a peptide corresponding
a 15 amino acid sequence specific to the  1D subunit of the L-type
calcium channel and was the kind gift of Drs. Nicole Martin-Moutot and
Michael Seager (Institut National de la Santé et de la Recherche
Médicale, Marseille). Polyclonal calcium channel antibodies
against peptides unique to either the 1A, 1B, or 1C subunits
were obtained from Alomone Labs (Jerusalem, Israel).
Light microscopic immunocytochemistry. Vertical retina
sections were prepared as described previously (Brandstätter et
al., 1996b). The sections were incubated for 1 hr in blocking solution [10% (vol/vol) normal goat serum (NGS), 1% (wt/vol) bovine serum albumin (BSA), 0.5% (vol/vol) Triton X-100 in PBS], followed by an
overnight incubation in the primary antibody diluted in incubation solution [3% (vol/vol) NGS, 1% (wt/vol) BSA, 0.5% (vol/vol) Triton X-100 in PBS]. The primary antibodies were diluted as follows: PMCA
(1:100), Na+/Ca2+ exchanger
(1:1000), synaptobrevin 2 (1:1,000), syntaxin 3 (1:50), D1a (1:1,000),
1A (1:60), 1B (1:200), and 1C (1:200). After they were washed,
the sections were incubated for 1 hr in the appropriate secondary
antibody diluted in incubation solution. The secondary antibodies were
diluted 1:1000 for CY3 conjugates and 1:50 for FITC conjugates. The
sections were washed again and then coverslipped with Mowiol
(Höchst, Frankfurt, Germany). Double-labeling experiments were
conducted similarly to single-labeling experiments except that both
primary antibodies were combined in the overnight incubation. After
they were washed, the sections were incubated with a mixture of
anti-sheep and anti-mouse secondary antibodies conjugated to CY3 and
FITC, respectively, and processed as for single-labeling experiments.
Photomicrographs were taken through an Axiophot microscope (Zeiss,
Germany). Confocal microscopic images were obtained with a Sarastro
2000 confocal laser scanning microscope system using a ×63/1.4 oil
immersion objective (Zeiss) as described elsewhere (Brandstätter
et al., 1996a).
Immunoblot. Total rat and tree shrew retinae were
solubilized in SDS sample buffer and equal quantities of protein were
separated by SDS-PAGE on a 10% (wt/vol) acrylamide gel (Laemmli,
1970). The proteins were electrophoretically transferred to
nitrocellulose and reacted with the PMCA antibody at a dilution of
1:100 as described previously (Brandstätter et al., 1996b).
Immunoreactivity was revealed with an ECL kit (Amersham, Braunschweig,
Germany) according to the manufacturer's instructions.
Electrophysiology. Two tree shrews (Tupaia
belangeri) were used for these experiments and were handled in
accordance with the National Institutes of Health guidelines for the
treatment of experimental animals. The animals were killed by an
overdose of sodium pentobarbital injected intraperitoneally. Retinas
were isolated, cut into four pieces and maintained in Ames medium
(Sigma). A piece of the retina was placed in the recording chamber and superfused with a control solution having the following composition (in
mM): NaCl 120, KCl 3.1, CaCl2 1.15, MgCl2 0.6, NaHCO3 23, and D-glucose
10. The pH of this solution was maintained at 7.4 by equilibration with
95% O2 and 5% CO2. The temperature was
23-25°C. Patch-clamp electrodes were filled with a solution having
the following composition (in mM): CsCl 125, Na+-HEPES 5, MgCl2 0.6, ATP 1, and GTP
0.1. A calcium buffer was not included, because we wanted to examine
the calcium buffering capacity of the surface membrane PMCA. Electrode
resistance was 6-10 M , before a whole-cell recording was
established. Series resistance was compensated for by at least 75%.
Even with series resistance compensation, voltage errors as large as 10 mV might have occurred, but this will not affect the conclusions we
draw from the results.
| |
RESULTS |
The PMCA is expressed in photoreceptor terminals
To determine whether the PMCA or the
Na+/Ca2+ exchanger is likely to
play a significant role in calcium extrusion from photoreceptor terminals, vertical sections of rat retina were immunostained with
antibodies specific for each of the two proteins (Fig.
1). The
Na+/Ca2+ exchanger antibody
showed weak staining throughout presumed cone photoreceptors and no
detectable staining of rods (Fig. 1A). The Na+/Ca2+ exchanger appeared to be
more abundant in the inner retina, where it strongly stained a
population of amacrine cell somata. Two immunoreactive bands were
visible in the inner plexiform layer (IPL), most probably originating
from the stained amacrine cells.
View larger version (70K):
[in this window]
[in a new window]
|
Figure 1.
Distribution of (A) the
Na+/Ca2+ exchanger and
(B) the PMCA in vertical sections of rat retina
detected by immunofluorescence microscopy. C, Nomarski
micrograph of one of the sections showing the retinal layers.
IS, Inner segments of photoreceptors;
OPL, outer plexiform layer; INL, inner
nuclear layer; IPL, inner plexiform layer;
GCL, ganglion cell layer. Scale bar, 40 µm.
|
|
The PMCA immunoreactivity was confined to the synaptic layers of the
retina (Fig. 1B). In contrast to the
Na+/Ca2+ exchanger, the PMCA
immunoreactivity was abundant in the outer plexiform layer (OPL), the
site of the photoreceptor synapses. The IPL was also stained, although
less intensely. The staining in the IPL suggests that bipolar cell
terminals also contain the PMCA, albeit at a much lower concentration
than the photoreceptor terminals. Within the IPL, two stronger bands of
staining could be seen that correspond to the cholinergic bands of
starburst amacrine cells (data not shown). The results from the
staining of the rat retina indicate that the PMCA is more abundant than the Na+/Ca2+ exchanger in the OPL
and thus more likely to be important for controlling calcium levels in
photoreceptor synaptic terminals.
In addition to rat, the PMCA was immunolocalized in the retinae
of the tree shrew and goldfish. The use of these three species allowed
a comparison between rod-dominated (rat) and cone-dominated (tree
shrew) mammalian retinae and between mammalian and fish retinae. The
tree shrew retina was chosen for further studies because the synaptic
terminals are large, an advantage for anatomical experiments, and the
photoreceptors are relatively easy to patch clamp, facilitating
physiological experiments. As in the rat retina, intense PMCA labeling
was seen in the OPL of both goldfish and tree shrew retina (Fig.
2B,D). The labeling
observed in the IPL of the tree shrew retina was similar to that
described above for the rat, including the two brighter bands (Fig.
2B), which were found to correspond to the
cholinergic bands (data not shown). The IPL of the goldfish retina was
labeled more weakly, and no stratification was observed (Fig.
2D). Faint but clear labeling was seen in the inner
IPL of the goldfish retina over large lobular structures, presumably
the giant axon terminals of the ON-bipolar cells. In all three species
examined, PMCA staining was restricted to the synaptic layers of the
retina and was strongest on the photoreceptor terminals in the OPL.
View larger version (101K):
[in this window]
[in a new window]
|
Figure 2.
Immunofluorescent staining of the PMCA in vertical
sections of tree shrew and gold fish retinae. A,
Nomarski micrograph of a vertical section of a tree shrew retina.
B, Immunofluorescent localization of the PMCA in the
tree shrew. C, Nomarski micrograph of a vertical section
of the gold fish retina. D, Immunofluorescent localization of the PMCA in gold fish. E,
High-magnification view of the PMCA staining in the OPL
of the tree shrew. F, High-magnification view of the
PMCA staining in the OPL of gold fish.
IS, Inner segments of photoreceptors;
OPL, outer plexiform layer; INL, inner
nuclear layer; IPL, inner plexiform layer;
GCL, ganglion cell layer. Scale bar (shown in
D for A-D): 40 µm; (shown in
F for E, F): 10 µm.
|
|
Inspection at high magnification of the PMCA immunoreactivity in the
OPL of both tree shrew and goldfish retinas revealed a striking pattern
of staining resembling a row of inverted "V" shapes (Fig.
2E,F). This pattern indicates that the PMCA is
restricted to the sides and neck of the photoreceptor terminal but is
absent from the base of the terminal, the presumed site of
neurotransmitter release.
The specificity of the PMCA antibody was determined with an immunoblot
of tree shrew and rat retinae (Fig. 3).
An immunoreactive band at 105 kDa was detected in the total retinal
proteins of both species, demonstrating that the antibody is highly
specific. The antigenic epitope has been mapped to the highly conserved hinge domain of the PMCA polypeptide (Adamo et al., 1992), and the
antibody recognizes PMCA isoforms in different tissues and cells (Borke
et al., 1990; Kessler et al., 1990; Magocsi and Penniston, 1991; de
Talamoni et al., 1993). The size of the band detected in the retina
suggests that the PMCA isoform present in retina is PMCA3, which is
found primarily in neural tissue (Stauffer et al., 1995). The
additional faint bands are likely to be other isoforms and degradation
products of the PMCA as have been described by others (Borke et al.,
1990; Kessler et al., 1990; de Talamoni et al., 1993).
View larger version (38K):
[in this window]
[in a new window]
|
Figure 3.
The PMCA antibody recognizes a protein of 105 kDa
in both rat (lane 1) and tree shrew (lane
2) retinae. Approximately equal quantities of total protein
from rat and tree shrew retinae were separated by SDS-PAGE and
immunoblotted with the PMCA antibody. The positions of size markers are
indicated on the left.
|
|
The PMCA is absent from the active zones of
photoreceptor terminals
To show the proximity of the PMCA to the synaptic active zones in
tree shrew photoreceptors, sections were double-labeled with the PMCA
antibody and antibodies against the synaptic plasma membrane protein
syntaxin 3 and the synaptic vesicle protein synaptobrevin, both of
which are associated with synaptic vesicle exocytosis from ribbon
synapses (Brandstätter et al., 1996b; Morgans et al., 1996).
Syntaxin 3 immunoreactivity (Fig.
4B) was concentrated at
the base of the terminal, marking the region where synaptic vesicles
fuse with the plasma membrane. The diffuseness of the band of syntaxin
3 immunoreactivity within the synaptic terminals suggests that syntaxin
3 is found on vesicles in addition to the plasma membrane, as has been
reported for SNAP-25, a syntaxin 3 binding protein (Brandstätter
et al., 1996b). The PMCA was found not to colocalize with syntaxin 3 (Fig. 4A,B), indicating that the PMCA is absent from
the active zones of photoreceptor synapses. In contrast to the syntaxin
3 labeling, the synaptobrevin antibody stained the photoreceptors from
the base of the terminal to the narrowing just beneath the soma (Fig.
4D), demonstrating that synaptic vesicles entirely
fill the photoreceptor terminals but are excluded from the soma. The
PMCA immunoreactivity mimics this compartmentalization, enclosing the
synaptobrevin staining from the base to the neck of the terminals (Fig.
4C,D).
View larger version (49K):
[in this window]
[in a new window]
|
Figure 4.
The PMCA is excluded from the sites of
neurotransmitter release in photoreceptor terminals. Vertical sections
of tree shrew retina were double-immunolabeled either with antibodies
against the PMCA and syntaxin 3 (A and B,
respectively) or with antibodies against the PMCA and synaptobrevin
(C and D, respectively). The scale in
A-D is the same as in Figure
2F.
|
|
We compared the localization of the PMCA to that of the
photoreceptor presynaptic calcium channels (Fig.
5). The distribution of the PMCA relative
to the calcium channels is of particular interest because the
arrangement of these proteins is likely to shape the calcium gradient
within the photoreceptor terminals. The calcium channels of
photoreceptor ribbon synapses have not been identified molecularly;
therefore we stained tree shrew retina sections with a panel of calcium
channel antibodies against peptide sequences unique to either the
1A, 1B, 1C, or 1D subunits. The 1A and 1B subunits
form calcium channels of the P/Q-type and N-type, respectively. Both
1C and 1D calcium channels are sensitive to dihydropyridines and
are thus of the L-type. Staining of the photoreceptor terminals was
observed with antibodies against both the 1D and 1B subunits;
however, only the 1D subunit appeared to be localized to the plasma
membrane, where it was confined to the base of the photoreceptor
terminals (Fig. 5). The 1B immunoreactivity appeared intracellular
(data not shown), similar to the synaptobrevin staining (Fig. 4).
Consistent with the absence of 1B from the plasma membrane,
 -conotoxin GVIA, a potent blocker of N-type channels, has been shown
not to inhibit photoreceptor calcium currents (Schmitz and Witkovsky,
1997). The significance of the apparent intracellular staining is
unclear at present. Antibodies against the 1A and 1C subunits did
not stain tree shrew photoreceptors (data not shown).
View larger version (111K):
[in this window]
[in a new window]
|
Figure 5.
Confocal image of the OPL in a vertical section of
tree shrew retina double-labeled for the PMCA
(red) and the calcium channel 1D subunit
(green). Scale bar, 5 µm.
|
|
Tree shrew retina sections were double-labeled with the PMCA antibody
and the antibody specific for the calcium channel 1D subunit (Fig.
5). The two antibodies stained strictly nonoverlapping domains within
the photoreceptor terminals. The L-type calcium channels were confined
to the base of the photoreceptor terminal, the presumed site of
glutamate release. The PMCA is excluded from this membrane domain but
is concentrated on the sides and neck of the terminal.
Calcium extrusion is dependent on the PMCA, but not on the
Na+/Ca2+exchanger
The identity of the calcium extrusion mechanism was confirmed
using an electrophysiological assay. Similar to other species (Maricq and Korenbrot, 1988; Barnes and Hille, 1989; Yagi and Macleish, 1994), tree shrew photoreceptors express a calcium-activated chloride conductance (Taylor and Morgans, 1998), and this was used to
monitor the relative intracellular calcium concentration (Roberts,
1993; Tucker and Fettiplace, 1996). Patch-clamp electrodes were applied
to the inner segments of the photoreceptors. Calcium currents were
activated by depolarizing pulses to  35 mV, from a holding potential
of 75 mV. When the chloride concentration of the intracellular
solution was equimolar with the external solution, large
calcium-activated chloride tail currents were observed after return to
the holding potential (Figs. 6, 7). These currents could be suppressed by replacing the extracellular calcium with cobalt (Fig. 6A), which is impermeant through
the channels, or with barium, which is permeant but does not activate
the chloride current (Taylor and Morgans, 1998). The time course of
these currents reflects the rate at which the intracellular calcium
concentration returns to resting levels. In 19 cells, the kinetics of
the calcium-activated chloride tail currents was measured ~1-2 min
after the whole-cell recording was established. In two cells, the time
course was well described by a single exponential function, with time
constants of 80 and 90 msec. In the remaining 17 cells a double
exponential function was required, with a fast time constant of 50 ± 25 msec and a slow time constant of 190 ± 80 msec. The
amplitude of the fast component was 1.8 times larger than the slow
component.
View larger version (12K):
[in this window]
[in a new window]
|
Figure 6.
Calcium extrusion is independent of
extracellular sodium. A, A 50 msec voltage pulse
activated a net inward calcium current. A large calcium-activated
chloride tail current is observed after repolarization. Replacement of
the extracellular calcium with cobalt (Co arrow)
completely suppressed the current during the pulse and the tail
current. Replacement of the extracellular sodium with lithium had no
effect on the time course of the tail current, indicating that calcium
extrusion was not dependent on
Na+/Ca2+ exchange activity.
B, The Ca Extrusion Index was unaffected
by either lithium or cobalt. Some points are missing during the cobalt application because the tail currents became too small for the Ca
extrusion to be calculated accurately.
|
|
If the photoreceptors contained an isoform of the
Na+/Ca2+ exchanger that was not
recognized by the antibody used in this study, it should become evident
in the physiological assay. Replacement of the extracellular sodium
with lithium is known to potently suppress
Na+/Ca2+ exchange activity
(Blaustein and Santiago, 1977), but this substitution had no effect on
the rate of decay of the calcium-activated chloride tail currents (Fig.
6A) or on the Ca Extrusion Index (Fig.
6B). Similar results were obtained in four cells.
This suggests that consistent with the staining, the
Na+/Ca2+ exchanger is
functionally absent from the photoreceptor terminals. The Ca Extrusion
Index provided a normalized measure for the efficiency of calcium
extrusion 300 msec after repolarization. It was calculated from the
tail currents as Ca Extrusion Index = 1 (tail current amplitude at 300 msec)/(peak tail current amplitude). For efficient extrusion this index will be unity, and for reduced extrusion it will tend toward zero.
Two pieces of evidence consistent with the strong PMCA staining in
photoreceptors are shown in Figure 7.
First, 1 mM Na+-orthovanadate, which is
known to block the PMCA but not the endoplasmic reticulum
Ca2+-ATPase in other systems (Carafoli, 1992),
potently increased the duration and magnitude of the calcium-activated
chloride tail currents (Fig. 7), even in the presence of 1 mM Mg2+-ATP. Second, the time course of
decay of the calcium-activated chloride tail currents was much slower
when Mg2+-ATP was not included in the intracellular
solution (Fig. 7B, open triangles). This is
consistent with the extrusion of calcium being dependent on ATPase
activity, because the intracellular ATP will be lost rapidly during the
course of the recording, leading to suppression of the PMCA activity.
After suppression of the PMCA activity, the tail currents lasted for
many seconds, rather than ~0.10 sec as was the case when the PMCA was
active.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 7.
PMCA activity is crucial for efficient calcium
extrusion from photoreceptors. A, The time course of the
calcium-activated chloride tail currents remained fairly stable during
a 4 min period (top records), but rapidly became large
and prolonged during a similar period when 1 mM
Na+-orthovanadate was included in the intracellular
solution (bottom records). B, The
open symbols accurately show that in the control cells
(1 mM Mg2+-ATP intracellular, 3 cells)
the Ca Extrusion Index remained stable for up to 10 min. The
closed circles show the effects of including 1 mM Na+-orthovanadate in the
intracellular solution (3 cells). A similar effect was observed when
Mg2+-ATP was omitted from the intracellular solution
(open triangles; 2 cells).
|
|
| |
DISCUSSION |
Photoreceptor terminals and other ribbon synapses are able to
support tonic neurotransmitter release, but to do so they are faced
with the challenge of maintaining a high calcium concentration at the
active zone while keeping calcium levels low elsewhere in the cell. Of
the two known means of calcium extrusion, the PMCA was shown by
immunofluorescence to be abundant in photoreceptor terminals, whereas
the Na+/Ca2+ exchanger was very
weakly detected in cones and not at all in rods. The PMCA antibody
intensely stained the OPL of both the rod-dominated retina of the rat
and the cone-dominated retina of the tree shrew, suggesting that the
PMCA is present in both rod and cone terminals. Using a
calcium-activated chloride current as an indicator of the intracellular
calcium concentration, we showed that inhibition of the PMCA
dramatically prolonged the chloride tail currents in tree shrew
photoreceptors. Inhibition of the
Na+/Ca2+ exchanger, on the other
hand, was without effect. The immunolocalization of the PMCA coupled
with the physiological data strongly suggest that PMCA activity is
critical for rapidly reducing the intracellular calcium concentration
in photoreceptor terminals.
Double-labeling of retina sections with antibodies against synaptic
markers and the PMCA demonstrated that the active zone is spatially
distinct from the sites of calcium efflux within the photoreceptor
terminals. Both syntaxin 3 and the calcium channel 1D subunit are
localized to the base of the synaptic terminal, the presumed site of
transmitter release, whereas the PMCA is concentrated along the sides
and neck of each terminal. The precise segregation of the PMCA and the
active zone markers raises the question of how photoreceptors are able
to establish and maintain these functionally and spatially distinct
plasma membrane domains within their synaptic terminals. Interactions
between membrane proteins and the submembrane cytoskeleton or
extracellular matrix are likely to be involved.
Of a panel of antibodies recognizing different calcium channel
1 subunits, only the 1D antibody appeared to stain the
photoreceptor plasma membrane. This is consistent with findings from
electrophysiological studies indicating the presence of L-type calcium
channels in ribbon synapses of photoreceptors (Rieke and Schwartz,
1994; Wilkinson and Barnes, 1996; Schmitz and Witkovsky, 1997),
bipolar cells (Heidelberger and Matthews, 1992; Tachibana et al., 1993;
Pan and Lipton, 1995), and inner ear hair cells (Art and Fettiplace, 1987). The relative distribution of the 1D calcium channels and the
PMCA indicates that the sites of calcium influx and efflux within a
photoreceptor synaptic terminal might be spatially segregated, possibly
giving rise to the formation of a standing calcium ion gradient across
the terminal (Fig. 8). This would be an
attractive mechanism to ensure high calcium levels required to sustain
continuous glutamate release from the terminal while preventing the
calcium concentration in the soma from rising and activating other
cellular processes. Although none of the other calcium channel
antibodies used in this study appeared to stain photoreceptor plasma
membranes in the tree shrew, the presence of other calcium channels in
the photoreceptor terminals cannot be ruled out. Intracellular calcium channels are also a possibility. Indeed, an intracellular staining pattern was observed in the photoreceptor terminals with an antibody against the 1B subunit of N-type calcium channels. The 1B
immunoreactivity may be caused by cross-reactivity with IP3
or Ca2+-gated intracellular calcium channels and
warrants further investigation.
View larger version (47K):
[in this window]
[in a new window]
|
Figure 8.
Diagram of the synaptic terminal of a tree shrew
cone. Sites of calcium influx and extrusion are segregated, possibly
giving rise to a standing calcium gradient in darkness when the cell is
depolarized. As depicted by a fusing synaptic vesicle, glutamate release occurs from the base of the terminal where the calcium channels
are located and the calcium concentration is highest.
|
|
The activity of the PMCA could influence the light response of bipolar
cells, the neurons postsynaptic to photoreceptors, particularly if
glutamate release from photoreceptors is controlled by the average
calcium concentration in the terminals rather than the high calcium
concentrations reached only within microdomains around open channels.
By measuring capacitance changes in response to photolysis of caged
calcium, Rieke and Schwartz (1996) found that a high rate of synaptic
vesicle exocytosis in rod terminals is maintained by a
Ca2+ concentration of 2-4 µm, which is the
average cytoplasmic Ca2+ concentration at the dark
resting potential. Bipolar cells, the other ribbon synapse-forming
neurons of the retina, have been shown to have two modes of glutamate
release: a transient mode that responds to calcium concentrations in
the range of 100 µm (Heidelberger et al., 1994; von Gersdorff and
Matthews, 1994), and a continuous mode supported by a rise in the
average calcium concentration of the terminal to 1 µm or less
(Lagnado et al., 1996).
If the rate of transmitter release is determined by the average
intracellular calcium concentration in photoreceptors, then the rate at
which the intracellular calcium concentration changes could limit the
response kinetics of the synaptic transfer. We have shown that
suppression of calcium extrusion greatly prolonged calcium-activated
chloride tail currents, indicating that intracellular calcium remained
elevated. What do the tail currents under control conditions tell us
about intracellular calcium concentration? It is tempting to suppose
that they track the intracellular free calcium, but alternatively they
may simply reflect the deactivation kinetics of the underlying chloride
channels. Unfortunately little is known about the kinetics or calcium
sensitivity of the calcium-activated chloride current in neurons, and
so we do not know how rapidly the current changes in response to
fluctuations in the calcium concentration. Because we saw
progressive prolongation of the tail currents, we can say, however,
that the tail currents provide an upper limit for the time constant of
calcium extrusion. This means that the calcium concentration dropped as
fast as, and not slower than, the 50 and 190 msec time constants
recorded. In this context it is intriguing to note that the fast time
constant is similar to that recorded for light-on responses in rat
bipolar cells (W. Taylor, unpublished results). This is consistent with the notion that the rate at which transmitter release from
photoreceptors can be shut off is determined by the rate at which the
intracellular calcium concentration can be lowered. If this is true,
then the PMCA, by determining the rate of calcium extrusion, could well exert a significant influence over the kinetics of synaptic transfer. In other systems the activity of the PMCA is subject to modulation by
direct interaction with calmodulin and by phosphorylation by protein
kinases A and C (Carafoli, 1992). Modulation of the photoreceptor PMCA
could add a further level of control over glutamate release and thus
synaptic transfer from these neurons.
| |
FOOTNOTES |
Received Sept. 10, 1997; revised Dec. 15, 1997; accepted Dec. 15, 1997.
We are grateful to Dr. Achim Kirsch for expert assistance with the
confocal microscopy.
Correspondence should be addressed to Dr. C. W. Morgans, Visual
Neurosciences, Division of Neuroscience, John Curtin School of Medical
Research, Canberra, ACT, 2600, Australia.
| |
REFERENCES |
-
Adamo H,
Caride A,
Penniston J
(1992)
Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.
J Biol Chem
267:14244-9[Abstract/Free Full Text].
-
Art JJ,
Fettiplace R
(1987)
Variation of membrane properties in hair cells isolated from the turtle cochlea.
J Physiol (Lond)
385:207-242[Abstract/Free Full Text].
-
Barnes S,
Hille B
(1989)
Ionic channels of the inner segment of tiger salamander cone photoreceptors.
J Gen Physiol
94:719-743[Abstract/Free Full Text].
-
Blaustein MP
(1988)
Calcium transport and buffering in neurons.
Trends Neurosci
11:438-443[Web of Science][Medline].
-
Blaustein MP,
Santiago EM
(1977)
Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons.
Biophys J
20:79-111[Web of Science][Medline].
-
Borke J,
Caride A,
Verma A,
Penniston J,
Kumar R
(1990)
Cellular and segmental distribution of Ca2(+)-pump epitopes in rat intestine.
Pflügers Arch
417:120-122[Web of Science][Medline].
-
Brandstätter JH,
Löhrke S,
Morgans CW,
Wässle H
(1996a)
Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, in the mammalian retina.
J Comp Neurol
370:1-10[Web of Science][Medline].
-
Brandstätter JH,
Wässle H,
Betz H,
Morgans CW
(1996b)
The plasma membrane protein SNAP-25, but not syntaxin, is present at photoreceptor and bipolar cell synapses in the rat retina.
Eur J Neurosci
8:823-828[Web of Science][Medline].
-
Carafoli E
(1992)
The Ca2+ pump of the plasma membrane.
J Biol Chem
267:2115-2118[Free Full Text].
-
Copenhagen DR,
Jahr CE
(1989)
Release of endogenous excitatory amino acids from turtle photoreceptors.
Nature
341:356-359.
-
de Talamoni N,
Smith C,
Wasserman R,
Beltramino C,
Fullmer C,
Penniston J
(1993)
Immunocytochemical localization of the plasma membrane calcium pump, calbindin-D28k, and parvalbumin in Purkinje cells of avian and mammalian cerebellum.
Proc Natl Acad Sci USA
90:11949-11953[Abstract/Free Full Text].
-
DeVries S,
Baylor DA
(1993)
Synaptic circuitry of the retina and olfactory bulb.
Cell
72:139-149.
-
DiPolo R,
Beauge L
(1979)
Physiological role of ATP-driven calcium pump in squid axon.
Nature
278:271-273[Medline].
-
Heidelberger R,
Matthews G
(1992)
Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons.
J Physiol (Lond)
447:235-256[Abstract/Free Full Text].
-
Heidelberger R,
Heinemann C,
Neher E,
Matthews G
(1994)
Calcium dependence of the rate of exocytosis in a synaptic terminal.
Nature
371:513-515[Medline].
-
Katz B,
Miledi R
(1967)
The timing of calcium action during neuromuscular transmission.
J Physiol (Lond)
189:535-544[Abstract/Free Full Text].
-
Kessler F,
Bennardini F,
Bachs O,
Serratosa J,
James P,
Caride A,
Gazzotti P,
Penniston J,
Carafoli E
(1990)
Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane.
J Biol Chem
265:16012-16019[Abstract/Free Full Text].
-
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
-
Lagnado L,
Cervetto L,
McNaughton PA
(1988)
Ion transport by the Na-Ca exchange in isolated rod outer segments.
Proc Natl Acad Sci USA
85:4548-4552[Abstract/Free Full Text].
-
Lagnado L,
Gomis A,
Job C
(1996)
Continuous vesicle cycling in the synaptic terminal of retinal bipolar cells.
Neuron
17:957-967[Web of Science][Medline].
-
Magocsi M,
Penniston J
(1991)
Oxytocin pretreatment of pregnant rat uterus inhibits Ca2+ uptake in plasma membrane and sarcoplasmic reticulum.
Biochim Biophys Acta
1063:7-14[Medline].
-
Maricq A,
Korenbrot J
(1988)
Calcium and calcium-dependent chloride currents generate action potentials in solitary cone photoreceptors.
Neuron
1:503-515[Web of Science][Medline].
-
Morgans C,
Brandstätter J,
Kellerman J,
Betz H,
Wässle H
(1996)
A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina.
J Neurosci
16:6713-6721[Abstract/Free Full Text].
-
Niggli V,
Adunyah E,
Penniston J,
Carafoli E
(1981)
Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids.
J Biol Chem
256:395-401[Abstract/Free Full Text].
-
Pan ZH,
Lipton SA
(1995)
Multiple GABA receptor subtypes mediate inhibition of calcium influx at rat retinal bipolar cell terminals.
J Neurosci
15:2668-2679[Abstract].
-
Reeves JP,
Hale CC
(1984)
The stoichiometry of the cardiac sodium-calcium exchange system.
J Biol Chem
259:7733-7739[Abstract/Free Full Text].
-
Rieke F,
Schwartz E
(1994)
A cGMP-gated current can control exocytosis at cone synapses.
Neuron
13:863-873[Web of Science][Medline].
-
Rieke F,
Schwartz E
(1996)
Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.
J Physiol (Lond)
493:1-8[Abstract/Free Full Text].
-
Roberts WM
(1993)
Spatial calcium buffering in saccular hair cells.
Nature
363:74-76[Medline].
-
Schmitz Y,
Witkovsky P
(1997)
Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel.
Neuroscience
78:1209-1216[Web of Science][Medline].
-
Stauffer TP,
Guerini D,
Carafoli E
(1995)
Tissue distribution of the four gene products of the plasma membrane Ca2+ pump.
J Biol Chem
270:12184-12190[Abstract/Free Full Text].
-
Tachibana M,
Okada T,
Arimura T,
Kobayashi K,
Piccolino M
(1993)
Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina.
J Neurosci
13:2898-2909[Abstract].
-
Taylor WR, Morgans CW (1998) Localization and properties of
voltage-gated calcium channels in cone photoreceptors of Tupaia
belangeri. Vis Neurosci, in press.
-
Tucker T,
Fettiplace R
(1996)
Monitoring calcium in turtle hair cells with a calcium-activated potassium channel.
J Physiol (Lond)
494:613-626[Abstract/Free Full Text].
-
von Gersdorff H,
Matthews G
(1994)
Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals.
Nature
367:735-739[Medline].
-
Wilkinson M,
Barnes S
(1996)
The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors.
J Gen Physiol
107:621-630[Abstract/Free Full Text].
-
Yagi T,
Macleish P
(1994)
Ionic conductances of monkey solitary cone inner segments.
J Neurophysiol
71:656-665[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1872467-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
H. Stohr, J. B. Heisig, P. M. Benz, S. Schoberl, V. M. Milenkovic, O. Strauss, W. M. Aartsen, J. Wijnholds, B. H. F. Weber, and H. L. Schulz
TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals
J. Neurosci.,
May 27, 2009;
29(21):
6809 - 6818.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Szikra, K. Cusato, W. B. Thoreson, P. Barabas, T. M. Bartoletti, and D. Krizaj
Depletion of calcium stores regulates calcium influx and signal transmission in rod photoreceptors
J. Physiol.,
October 15, 2008;
586(20):
4859 - 4875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Lee, A. Jimenez, G. Cui, and F. Haeseleer
Phosphorylation of the Ca2+-Binding Protein CaBP4 by Protein Kinase C {zeta} in Photoreceptors
J. Neurosci.,
November 14, 2007;
27(46):
12743 - 12754.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. R. MacLeish and C. A. Nurse
Ion Channel Compartments in Photoreceptors: Evidence From Salamander Rods With Intact and Ablated Terminals
J Neurophysiol,
July 1, 2007;
98(1):
86 - 95.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Yang, B. Pawlyk, X.-H. Wen, M. Adamian, M. Soloviev, N. Michaud, Y. Zhao, M. A. Sandberg, C. L. Makino, and T. Li
Mpp4 is required for proper localization of plasma membrane calcium ATPases and maintenance of calcium homeostasis at the rod photoreceptor synaptic terminals
Hum. Mol. Genet.,
May 1, 2007;
16(9):
1017 - 1029.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. P. Jensen, A. G. Filoteo, T. Knopfel, and R. M. Empson
Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3
J. Physiol.,
February 15, 2007;
579(1):
85 - 99.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Maeda, J. Lem, K. Palczewski, and F. Haeseleer
A Critical Role of CaBP4 in the Cone Synapse
Invest. Ophthalmol. Vis. Sci.,
November 1, 2005;
46(11):
4320 - 4327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Mansergh, N. C. Orton, J. P. Vessey, M. R. Lalonde, W. K. Stell, F. Tremblay, S. Barnes, D. E. Rancourt, and N. T. Bech-Hansen
Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina
Hum. Mol. Genet.,
October 15, 2005;
14(20):
3035 - 3046.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-H. Kim, N. Korogod, R. Schneggenburger, W.-K. Ho, and S.-H. Lee
Interplay between Na+/Ca2+ Exchangers and Mitochondria in Ca2+ Clearance at the Calyx of Held
J. Neurosci.,
June 29, 2005;
25(26):
6057 - 6065.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Zhang, A. Beuve, and E. Townes-Anderson
The Nitric Oxide-cGMP Signaling Pathway Differentially Regulates Presynaptic Structural Plasticity in Cone and Rod Cells
J. Neurosci.,
March 9, 2005;
25(10):
2761 - 2770.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Cia, A. Bordais, C. Varela, V. Forster, J. A. Sahel, A. Rendon, and S. Picaud
Voltage-Gated Channels and Calcium Homeostasis in Mammalian Rod Photoreceptors
J Neurophysiol,
March 1, 2005;
93(3):
1468 - 1475.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Lipscombe, T. D. Helton, and W. Xu
L-Type Calcium Channels: The Low Down
J Neurophysiol,
November 1, 2004;
92(5):
2633 - 2641.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Qu, R. W. Wei, W. Mann, and H. C. Hartzell
Two Bestrophins Cloned from Xenopus laevis Oocytes Express Ca2+-activated Cl- Currents
J. Biol. Chem.,
December 5, 2003;
278(49):
49563 - 49572.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hirasawa and A. Kaneko
pH Changes in the Invaginating Synaptic Cleft Mediate Feedback from Horizontal Cells to Cone Photoreceptors by Modulating Ca2+ Channels
J. Gen. Physiol.,
November 24, 2003;
122(6):
657 - 671.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Koschak, D. Reimer, D. Walter, J.-C. Hoda, T. Heinzle, M. Grabner, and J. Striessnig
Cav1.4{alpha}1 Subunits Can Form Slowly Inactivating Dihydropyridine-Sensitive L-Type Ca2+ Channels Lacking Ca2+-Dependent Inactivation
J. Neurosci.,
July 9, 2003;
23(14):
6041 - 6049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kreft, D. Krizaj, S. Grilc, and R. Zorec
Properties of Exocytotic Response in Vertebrate Photoreceptors
J Neurophysiol,
July 1, 2003;
90(1):
218 - 225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. L. Limke, J. K. L. Otero-Montanez, and W. D. Atchison
Evidence for Interactions between Intracellular Calcium Stores during Methylmercury-Induced Intracellular Calcium Dysregulation in Rat Cerebellar Granule Neurons
J. Pharmacol. Exp. Ther.,
March 1, 2003;
304(3):
949 - 958.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Hurtado, S. Borges, and M. Wilson
Na+-Ca2+ Exchanger Controls the Gain of the Ca2+ Amplifier in the Dendrites of Amacrine Cells
J Neurophysiol,
November 1, 2002;
88(5):
2765 - 2777.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. T. Blackwell
Calcium Waves and Closure of Potassium Channels in Response to GABA Stimulation in Hermissenda Type B Photoreceptors
J Neurophysiol,
February 1, 2002;
87(2):
776 - 792.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Zhong, V. Beaumont, and R. S. Zucker
Roles for Mitochondrial and Reverse Mode Na+/Ca2+ Exchange and the Plasmalemma Ca2+ ATPase in Post-Tetanic Potentiation at Crayfish Neuromuscular Junctions
J. Neurosci.,
December 15, 2001;
21(24):
9598 - 9607.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. L. Fain, H. R. Matthews, M. C. Cornwall, and Y. Koutalos
Adaptation in Vertebrate Photoreceptors
Physiol Rev,
January 1, 2001;
81(1):
117 - 151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Oberwinkler and D. G. Stavenga
Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors
PNAS,
July 18, 2000;
97(15):
8578 - 8583.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Koulen, R. Kuhn, H. Wassle, and J. H. Brandstatter
Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor
PNAS,
August 17, 1999;
96(17):
9909 - 9914.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Koulen, E. L. Fletcher, S. E. Craven, D. S. Bredt, and H. Wassle
Immunocytochemical Localization of the Postsynaptic Density Protein PSD-95 in the Mammalian Retina
J. Neurosci.,
December 1, 1998;
18(23):
10136 - 10149.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
