1 Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, D-52425 Jülich, Germany; and 2 Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Na+/Ca2+
exchange has been investigated in squid
(Loligo
pealei) rhabdomeric membranes.
Ca2+-containing vesicles have been
prepared from purified rhabdomeric membranes by extrusion through
polycarbonate filters of 1-µm pore size. After removal of external
Ca2+, up to 90% of the entrapped
Ca2+ could be specifically
released by the addition of Na+;
this finding indicates that most of the vesicles contained
Na+/Ca2+
exchanger. The Na+-induced
Ca2+ efflux had a half-maximum
value (K1/2) of
~44 mM and a Hill coefficient of ~1.7. The maximal
Na+-induced
Ca2+ efflux was ~0.6 nmol
Ca2+ · s1 · mg
protein
1. Similar
Na+-induced
Ca2+ effluxes were measured if
K+ was replaced with
Li+ or
Cs+. Vesicles loaded with
Ca2+ by
Na+/Ca2+
exchange also released this Ca2+
by
Na+/Ca2+
exchange, suggesting that
Na+/Ca2+
exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of
Ca2+ efflux enhanced by
approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin,
Na+/Ca2+
exchange was characterized by a
K1/2 of ~25 mM
and a Hill coefficient of 1.6. For these vesicles, the maximal
Na+-induced
Ca2+ efflux was about twice as
great as in control vesicles. We conclude that
Na+/Ca2+
exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.
photoreceptor; invertebrate; calcium extrusion; calcium homeostasis
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INTRODUCTION |
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INTRACELLULAR Ca2+ (Ca2+i) plays important roles in the physiology of invertebrate rhabdomeric photoreceptors. For example, Ca2+i has been found to mediate light adaptation in the photoreceptors of Limulus; i.e., light causes a rise in Ca2+i concentration (6, 7) and a rise in Ca2+i concentration causes a reduction in light sensitivity (9, 20, 21). Moreover, Ca2+i has been proposed to participate in the excitation of these photoreceptors, either as a mediator (30, 38) or as a modulator (5) of a step in the cascade of reactions in phototransduction. Therefore, the regulation of the concentration of Ca2+i in these photoreceptors has important consequences for their functions. The concentration of Ca2+i depends on the entry of Ca2+ into the cytosol (from both influx through plasma membrane channels and release from intracellular stores) and on the removal of Ca2+ from the cytosol (by both sequestration into those stores and extrusion through the plasma membrane). As in other cells, the mechanisms for extrusion of Ca2+ through the plasma membrane probably include both a high-affinity Ca2+ pump (25) and a low-affinity, high-capacity Na+/Ca2+ exchange mechanism similar to that described for other cells (19).
Na+/Ca2+ exchangers have been found in a wide variety of cells and have been found to differ in several properties. One type of exchanger is typified by that found in vertebrate retinal rods, and another is typified by that found in vertebrate cardiac muscle. These two types differ markedly in their amino acid sequences (for rods and rat brain, see Refs. 34 and 37, respectively; for cardiac muscle, see Refs. 26 and 27). The families also differ in their sensitivities to the presence of K+; the rod exchanger both transports and requires K+ (10, 31, 36) and operates with a stoichiometry of 4 Na+:1 Ca2+ plus 1 K+, whereas the cardiac muscle exchanger is insensitive to K+ and operates with a stoichiometry of 3 Na+:1 Ca2+ (13, 26, 33). In addition, the Na+/Ca2+ exchanger in squid giant axons has been extensively studied by electrophysiological and radiotracer techniques; it has been found that this exchanger has a stoichiometry of 3 Na+:1 Ca2+ and does not transport K+ (3, 4, 12).
Several electrophysiological studies of invertebrate photoreceptors (1, 14, 21-23) have shown that if the Na+ gradient across the plasma membrane is reduced in the presence of extracellular Ca2+, then the sensitivity of the photoreceptor is reduced. Because it is known that an increase in Ca2+i causes a decrease in sensitivity (9, 20, 21), these studies suggest that there is a rise in Ca2+i concentration due to reduced Na+/Ca2+ exchange. Using aequorin as an indicator for Ca2+i, O'Day et al. (28, 29) demonstrated directly that the Ca2+i concentration rose after a reduction of the Na+ gradient, either by injection of Na+ or by lowering the concentration of extracellular Na+. Taken together, these findings suggest that there is a Na+/Ca2+ exchanger in some region of the plasma membrane of Limulus photoreceptors.
The retina of the squid comprises almost entirely photoreceptor cells (11). The rhabdomeric membranes from the squid retina can be prepared simply, almost in isolation, from frozen retinas (18, 35). Thus the squid retina allows the straightforward preparation of vesicles from rhabdomeric membranes for the study of Ca2+ fluxes. In this study, we have used this technique to demonstrate that the rhabdomeric membranes of the squid (Loligo pealei) photoreceptor contain a Na+/Ca2+ exchanger that is distinctly different from the Na+/Ca2+-K+ exchanger of vertebrate photoreceptors.
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MATERIALS AND METHODS |
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Dissection of squid retinas. Eyes were removed from squid (L. pealei) collected at the Marine Biological Laboratory (Woods Hole, MA). Animals were held in oxygenated seawater in total darkness for not less than 1 h before dissection. Retinas were dissected under infrared illumination with the aid of image converters. Alternatively, animals were held in large tanks of running seawater after nightfall, and retinas were dissected under dim white light. In both cases, the screening pigment within the retinas was found in its dark-adapted position at the base of the rhabdomeres. The retinas were dissected free from the sclera in oxygenated seawater, rinsed in Ca2+-free seawater, and rapidly frozen by immersion either in liquid nitrogen or in isopentane at liquid nitrogen temperature.
Purification of rhabdomeric membranes. A piece of frozen retina was thawed in homogenization buffer (0.5 ml/half-retina) that contained (in mM) 200 KCl, 1 MgCl2, 0.1 EDTA, 0.1 dithiothreitol, 10 HEPES adjusted to pH 7.4 with KOH, and the protease inhibitors aprotinin and leupeptin (5 µg/ml each). The piece of retina was slowly thawed while keeping the buffer cooled on ice. After ~1 h, deep red clumps of the rhabdomeric membranes dislodged readily from the retina after slight agitation of the buffer. The remainder of the retina, which contained the bulk of the screening pigment, was removed, and the rhabdomeric membranes were pelleted by centrifugation at 5,000 g for 5 min. The pellet was homogenized with a plastic pestle, resuspended in homogenization buffer, and layered onto a cushion of 40% sucrose in homogenization buffer (minus the protease inhibitors). The sample was centrifuged at 18,300 g at 4°C for 60 min. The pellet containing most of the remaining screening pigment was discarded. The mat of rhabdomeric membranes was collected from above the sucrose cushion, resuspended in homogenization buffer, and centrifuged at 18,300 g for 10 min.
Purified rhabdomeric membranes, i.e., membranes free of screening pigment granules, were obtained by repeated hypotonic washing as follows. The membrane pellet was gently homogenized with a plastic pestle in a 1.5-ml plastic centrifuge tube and resuspended in ice-cold hypotonic buffer that contained 0.5 mM HEPES and 1 mM EDTA adjusted to pH 7.4 with Tris base. The membranes were centrifuged for 15 min at 18,300 g and 4°C. The tubes then contained a dark solid pellet; on top of the pellet was a fluffy red layer composed of rhabdomeric membranes. The rhabdomeric membranes were resuspended by slight agitation and collected. Those that remained within the pellet could eventually be separated from the screening pigment by repeated washing in hypotonic buffer. The total yield of rhabdomeric membranes was ~650 µg protein/retina after 8 cycles of hypotonic washes. In some experiments, the protocol for preparing membranes was modified in the following ways. After being collected from the sucrose step-gradient centrifugation, the membranes were resuspended in ice-cold hypotonic buffer that contained (in mM) 0.5 HEPES, 1 MgCl2, and 1 EDTA adjusted to pH 7.4 with Tris base. The membranes were centrifuged, and the fluffy pink layer was collected after very gentle agitation. Most of the remaining membranes were collected by one further resuspension and centrifugation in the hypotonic buffer. These membranes were pooled and centrifuged as described above. The resulting pellet was resuspended in homogenization buffer and stored on ice. This modification of the protocol reduced any effects of prolonged washing of the membranes in hypotonic buffer.Preparing vesicles from rhabdomeric membranes. Freshly prepared purified rhabdomeric membranes were resuspended in loading buffer that contained (if not noted otherwise; in mM) 100 KCl, 10 HEPES, 10 CaCl2, and 1 dithiothreitol adjusted to pH 7.4 with Tris base. Vesicles were formed by forcing the squid rhabdomeric membranes once through a polycarbonate filter with a pore size of 3 µm backed with a polycarbonate filter with a pore size of 1 µm (Nuclepore; Costar, Cambridge, MA). Ca2+ not included was removed from preparations of vesicles by batch treatment with iminodiacetic acid chelating resin in the K+ form (Chelex 100, mesh 50-100; Bio-Rad) equilibrated to pH 7.4 in the measurement buffer. The measurement buffer contained (if not noted otherwise; in mM) 100 KCl, 10 HEPES, and 1 dithiothreitol adjusted to pH 7.4 with Tris base.
To investigate the influence of K+ on Na+/Ca2+ exchange, vesicles were prepared from rhabdomeric membranes in the complete absence of K+ by replacement of KCl by LiCl in all solutions. In these solutions, the pH of the HEPES buffer was adjusted with LiOH. These vesicles were loaded with Ca2+ by extrusion in 10 mM CaCl2, 100 mM LiCl (or 100 mM CsCl), and 10 mM HEPES adjusted to pH 7.4 with LiOH. The Ca2+ not included was removed by batch treatment of the vesicles with Chelex resin in the Li+ or Tris+ form. To investigate Na+-induced Ca2+ influx into vesicles, rhabdomeric membranes were extruded in 100 mM NaCl buffered in 10 mM HEPES-Tris (or HEPES-LiOH; pH 7.4) and diluted 1:5 with 100 mM KCl (or, for K+-free experiments, LiCl or CsCl) plus 10 mM CaCl2 in the same buffer. Thus a Na+ outward gradient and a Ca2+ inward gradient were generated. Ca2+ uptake was terminated after a chosen time by removal of external Ca2+ with Chelex resin (in the K+ form or, for K+-free experiments, in the Li+ or Tris+ form).Measuring Ca2+ effluxes from rhabdomeric membrane vesicles. Ca2+-indicating dye arsenazo III (2, 8) was added to the vesicle suspension (to a final concn of 50 µM), 1 ml was pipetted into a stirring cuvette with a 10-mm-long light path (119.004-OS; Hellma, Müllheim, Germany), and the cuvette was placed into a dual-wavelength spectrophotometer (Aminco DW-2000; SLM Instruments, Urbana, IL). The spectrophotometer was equipped with a cuvette-stirring motor (model 333; Hellma) to allow rapid mixing within the cuvette. The temperature of the cuvette was kept constant at 4°C; a continuous flush of nitrogen (4.5 l/min) was directed at the optical surfaces of the cuvette to avoid condensation of moisture. The difference in absorbance between 652 and 700 nm vs. time was recorded (dual-wavelength mode of the spectrophotometer). Additions were made with Hamilton (Bonaduz, Switzerland) pipettes through a light-tight inlet fitting. After each measurement, the suspensions were recovered and the total protein content was determined by the amido black procedure (17) using Millipore HA filters with a 0.45-µm pore size (Millipore, Bedford, MA).
Millimolar concentrations of alkali ions decrease the apparent affinity of arsenazo III for Ca2+ (2), as indicated by the downward deflections of the absorbance signal on the additions of K+ and Na+ (Fig. 1). Ca2+ effluxes were determined by linear regression of the data from 10-20 s after each addition (to ensure complete mixing) and corrected for the Ca2+ efflux measured immediately before the addition (see Fig. 2). Ca2+ concentration changes were calibrated within the cuvette in each experiment by using the changes in absorbance produced by two successive additions of CaCl2 (in increments of 2 µM). To compare different experiments, the Ca2+ effluxes were divided by the total protein mass present in the cuvette. Ca2+ efflux from vesicles was also measured with the fluorescent Ca2+ indicator fluo 3 (15, 24). Ca2+-loaded vesicles were prepared by extrusion as described above and placed in a quartz stirring cuvette (109.004F-QS; Hellma), with fluo 3 added to 10 µM. The measurements were made in a fluorometer (model 750; McPherson, Acton, MA) fitted with a temperature-controlled cuvette chamber (held at 5°C) and a magnetic stirrer (model 333; Hellma). The excitation wavelength was 480 nm (band width 16 nm), and emission above 520 nm was detected with a high-pass filter (KV520; Schott, Mainz, Germany). Additions to the cuvette were made with Hamilton syringes through a light-tight fitting. Ca2+ concentration changes were calibrated within the cuvette by known additions of CaCl2 at the end of each experiment.Limited proteolysis of rhabdomeric membranes. The influence of mild trypsin treatment on Na+-induced Ca2+ effluxes was investigated. After preparation of the vesicles by extrusion in Ca2+ loading buffer, 2 µg/ml bovine pancreas trypsin (type XIII; Sigma) were added to the suspension at 4°C for various periods (up to 30 min; see Fig. 8). The vesicles were used for Ca2+ efflux experiments after termination of the proteolysis by addition of 40 µg/ml of soybean trypsin inhibitor (Sigma).
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RESULTS |
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Ca2+ efflux from
Ca2+-loaded vesicles prepared by
extrusion of purified rhabdomeric membranes was measured with
Ca2+ indicator dye arsenazo III at
4°C. A low temperature was necessary because squid rhabdomeric
membranes deteriorate rapidly at temperatures >10°C. There was a
passive Ca2+ efflux from the
vesicles of ~1.8 nmol
Ca2+ · min1 · mg
protein
1 (Fig.
1). We do not know the origin
of this leakiness of the vesicles; the
Ca2+-channel blockers
D-cis-diltiazem,
nifedipine, and gallopamil did not reduce the passive
Ca2+ efflux (data not shown). The
addition of KCl (to a final concn of 95 mM) elicited a downward
deflection of the optical signal due to a decrease in the apparent
Ca2+ affinity of
Ca2+ indicator arsenazo III (2)
but did not change the passive Ca2+ efflux. However, the addition
of NaCl (to a final concn of 90 mM) elicited an almost 20-fold increase
in Ca2+ efflux, which resulted in
a release of most of the entrapped Ca2+ after 5 min. The addition of
calcium ionophore A-23187 (to a concn of 2.5 µM) released virtually
all of the Ca2+; a second addition
of A-23187 yielded little further
Ca2+ efflux. Changes in
Ca2+ concentration were calibrated
at the end of the experiment with two consecutive additions of
CaCl2 in increments of
concentration of 2 µM. For vesicles prepared from membranes that were
subjected to minimal washes in hypotonic buffer, up to 90% of the
total included Ca2+ was released
by the addition of NaCl. Our findings suggest that most of the vesicles
contain
Na+/Ca2+
exchange proteins.
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The sum of Ca2+ effluxes elicited
by consecutive additions of Na+
and ionophore A-23187 was approximately constant (Fig.
2) and was ~91 nmol
Ca2+/mg protein in this example;
for the whole set of different Na+
additions (as shown in Fig. 4) the sum of
Na+- and ionophore-induced
Ca2+ releases was 89.2 ± 13.4 nmol Ca2+/mg protein
(n = 8). This finding demonstrates
that the Na+-releasable
Ca2+ pool is A-23187 sensitive.
The recordings in Fig. 2 have been rescaled by using the
Ca2+ calibrations at the end of
each recording (not shown) to plot directly the amounts of released
Ca2+ per amount of protein. The
addition of FCCP (5 µM) or valinomycin (5 µM) did not measurably
influence the Na+-induced
Ca2+ efflux (data not shown). No
Na+-induced
Ca2+ efflux could be elicited
after an A-23187-induced Ca2+
efflux (data not shown).
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Ca2+ efflux was also measured with
fluorescent Ca2+ indicator fluo 3. An initial passive efflux was dramatically increased after the addition
of NaCl (Fig.
3A). The
addition of the nonfluorescent ionophore ionomycin elicited some
additional efflux; a subsequent addition of Triton X-100 elicited
minimal further efflux. Figure 3B
shows that the Ca2+ efflux was not
elicited by the addition of an equal amount of KCl to the cuvette. All
the experiments that we repeated with fluo 3 were entirely consistent
with those with arsenazo III. Therefore, because the two indicators are
chemically so different, it is unlikely that there are any appreciable
pharmacological effects of arsenazo III present in our measurements. In
addition, we prepared vesicles from rhabdomeric membranes from living,
unfrozen squid retinas. Measurements of
Ca2+ efflux made with fluo 3 from
these vesicles from fresh retinas (data not shown) were not appreciably
different from those made with vesicles from frozen retinas.
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To determine the relationship between the
Ca2+ efflux and the concentration
of Na+ outside the vesicles,
the Na+-induced
Ca2+ efflux was calculated as the
difference between the slopes of the curves of the change in
absorbance of arsenazo III vs. time before and 10 s after the addition
of Na+ (see Fig. 2). These values
of Ca2+ efflux were divided by the
mass of total protein present in each Ca2+ release experiment. Figure
4 shows the
Na+ dependence of the
Ca2+ efflux, as
determined in one experiment. In four similar experiments, the
maximal Ca2+ efflux was 0.54 ± 0.17 nmol
Ca2+ · s1 · mg
protein
1, the half-maximum
value (K1/2)
was 44.0 ± 6.0 mM, and the Hill coefficient was 1.73 ± 0.21.
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The
Na+/Ca2+
exchanger of the cardiac sarcolemma is functionally active in the
absence of K+. However, in
vertebrate photoreceptors, K+ is
cotransported with Ca2+ and little
or no Ca2+ is transported in the
absence of K+ (26). We
investigated the influence of K+
on
Na+/Ca2+
exchange in vesicles of the rhabdomeric membranes from squid retinas.
Figure 5 shows that the
Na+/Ca2+
exchange rate does not change appreciably if
K+ is replaced with
Li+ or
Cs+. Similarly, substitution of
K+ with tetramethylammonium or
Tris did not markedly influence the rate of
Na+/Ca2+
exchange; however, the rate of
Na+/Ca2+
exchange was significantly lower if
K+ was replaced with
N-methylglucamine (data not shown).
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Na+/Ca2+
exchange in vesicles of squid rhabdomeric membranes was also detected
as Na+-dependent
Ca2+ influx. Vesicles were
prepared to include no Ca2+ by
extrusion, either in 100 mM NaCl or in 100 mM KCl, without any added
Ca2+. Before the experiment, 10 mM
Ca2+ was added to the outside of
the vesicles for a chosen duration and then removed by ion-exchange
resin. Ca2+ uptake was monitored
as Ca2+ released upon the addition
of A-23187 (to a concn of 2.5 µM). Figures
6 and 7 show
that vesicles containing Na+ take
up Ca2+ markedly more than
vesicles containing K+, in
particular for short incubations (Fig. 7), yet there is also a clear
Ca2+ uptake in the absence of
Na+ due to passive
Ca2+ leakage. There is no
significant difference in Ca2+
uptake if K+ is replaced by
Li+ (Fig. 6). Moreover, the
Na+-induced
Ca2+ flux is greater for the
vesicles loaded by
Na+/Ca2+
exchange than for the
K+-containing vesicles that
entrapped Ca2+ only by passive
leakage (Fig. 7). The latter observation is consistent with the ability
of the
Na+/Ca2+
exchanger to transport Ca2+ in
both the forward and reverse exchange modes (see
DISCUSSION).
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It has been reported that the cardiac sarcolemmal
Na+/Ca2+
exchanger is activated by mild proteolysis (32). Therefore, we
investigated Na+/Ca2+
exchange after mild trypsin treatment of vesicles prepared from squid
rhabdomeric membranes. Very mild trypsin treatment caused a conspicuous
increase of the Ca2+ efflux
elicited after the addition of Na+
(by up to a factor of ~6 at 95 mM
Na+; Fig.
8). With 2 µg/ml trypsin at 4°C, the
half-maximal activation was reached after 3.8 min of proteolysis.
Titration of the
Na+/Ca2+
exchange with Na+ yielded an
approximately twofold smaller
K1/2 for
trypsin-treated vesicles than for non-trypsin-treated ones and a
twofold enhancement of the maximal
Na+/Ca2+
exchange rate at the saturating
Na+ concentration (data not
shown).
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Finally, Na+/Ca2+ exchange is rather specific for Ca2+. If Ca2+ is replaced with Sr2+, the maximal exchange rate drops by roughly a factor of two. Little or no release is observed if Ca2+ is replaced with Ba2+ (data not shown).
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DISCUSSION |
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In this study, we demonstrate directly that squid rhabdomeric membranes contain a Na+/Ca2+ exchanger. We find that the addition of Na+ releases up to 90% of the Ca2+ entrapped in vesicles made from rhabdomeric membranes that have had minimal exposure to hypotonic solutions. This finding suggests that most of these vesicles contain a Na+/Ca2+ exchanger. The half-maximal Na+-induced Ca2+ efflux occurred at ~45 mM Na+. The Hill coefficient of ~1.7 indicates that more than one Na+ was transported per Ca2+. As a comparison, the Na+/Ca2+ exchanger in vertebrate rods has a K1/2 of 67 mM and a Hill coefficient of 2.0 (16).
K+ is obligatory for Na+/Ca2+ exchange in vertebrate photoreceptors, but not for Na+/Ca2+ exchange in the cardiac sarcolemma (26). This striking functional difference in the ion requirement of Na+/Ca2+ exchange is also reflected in the amino acid sequences; the sequences for the two proteins are almost unrelated (34). We report here that Na+/Ca2+ exchange across squid rhabdomeric membranes does not depend obligatorily on K+. This result indicates that this Na+/Ca2+ exchanger presumably resembles the cardiac Na+/Ca2+ exchangers more than the exchanger in vertebrate rods.
Na+/Ca2+ exchangers not only can extrude Ca2+ from the cytoplasm to the extracellular space (forward mode) but also can mediate Ca2+ influx into cells if the Na+ gradient is experimentally inverted (reverse mode). We used vesicles that had been Ca2+ loaded by Na+/Ca2+ exchange to show that the Na+/Ca2+ exchanger in squid rhabdomeric membranes can work in both the forward and reverse modes. Our preparations most likely have both inside-out and right-side-out vesicles. The Na+-induced increase in Ca2+ uptake might be mediated by either mode of Na+/Ca2+ exchange, depending on the sidedness of each particular vesicle. For inside-out vesicles with high inside Na+ concentration, if Ca2+ had been entrapped solely by forward-mode Na+/Ca2+ exchange, then a reversal of the Na+ gradient by the addition of Na+ outside of the vesicle could only elicit a Ca2+ release from these inside-out vesicles by the reverse mode of Na+/Ca2+ exchange. Similarly, right-side-out vesicles loaded by reverse-mode Na+/Ca2+ exchange would be emptied by forward-mode exchange. This means that the Na+-enhanced Ca2+ uptake would then lead to an increased Na+-induced Ca2+ efflux only if both modes of Na+/Ca2+ exchange can be active. Figure 7 shows that the Ca2+ efflux is markedly increased for vesicles loaded with Ca2+ compared with vesicles loaded only by passive Ca2+ influx. We infer from this result that the Na+/Ca2+ exchanger in vesicles prepared from squid rhabdomeric membranes can operate in both forward and reverse modes, as do all other Na+/Ca2+ exchangers.
Mild protease treatment stimulates the Na+/Ca2+ activity of both cardiac and rod Na+/Ca2+ exchangers. The same feature was also observed after mild trypsin treatment of the squid rhabdomeric Na+/Ca2+ exchanger. Proteolysis not only enhanced the maximal turnover by almost exactly a factor of two but also decreased the K1/2 for Na+ by a factor of about two. Therefore, stimulation of Na+/Ca2+ exchange by mild proteolysis was particularly marked for nonsaturating Na+ concentrations (Fig. 8). Proteolytic removal of an inhibitory domain of the exchanger may account for this stimulatory effect, although we cannot exclude the proteolytic degradation of an auxiliary protein.
In summary, we report strong evidence that squid rhabdomeric membranes contain Na+/Ca2+ exchange proteins with the following characteristics: 1) Na+ cooperatively activates the exchange; 2) Na+/Ca2+ exchange does not depend on K+; 3) Na+/Ca2+ exchange is strongly stimulated by mild proteolysis; and 4) both forward and reverse modes of exchange exist. We conclude that, similar to what is found for vertebrate photoreceptors, Na+/Ca2+ exchange participates in Ca2+ homeostasis in invertebrate photoreceptors. However, the K+ outward gradient is not used for Na+/Ca2+ exchange in photoreceptors of squid as it is for vertebrate photoreceptors.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. De Weer, U. B. Kaupp, and E. Nasi for valuable discussions and H. Erkens and H. D. Grammig for technical help.
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FOOTNOTES |
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This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ba 721/1-2) to P. J. Bauer, by National Eye Institute Grant EY-05166 to J. E. Brown, and in part by a grant to the Dept. of Ophthalmology, Albert Einstein College of Medicine, from the Research to Prevent Blindness Foundation, New York, NY.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. J. Bauer, Institut für Biologische Informationsverarbeitung, P.O. Box 1913, Forschungszentrum Jülich, D-52425 Jülich, Germany (E-mail: P.J.Bauer{at}fz-juelich.de).
Received 8 May 1998; accepted in final form 1 December 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bader, C. R.,
F. Baumann,
and
D. Bertrand.
Role of intracellular calcium and sodium in light adaptation in the retina of the honey bee drone (Apis mellifera, L.).
J. Gen. Physiol.
67:
475-491,
1976[Abstract].
2.
Bauer, P. J.
Affinity and stoichiometry of calcium binding by arsenazo III.
Anal. Biochem.
110:
61-72,
1981[Medline].
3.
Beaugé, L.,
and
R. DiPolo.
Effects of monovalent cations on Na-Ca exchange in nerve cells.
Ann. NY Acad. Sci.
639:
147-155,
1991[Medline].
4.
Blaustein, M. P.
Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons.
Biophys. J.
20:
79-111,
1977[Abstract].
5.
Bolsover, S. R.,
and
J. E. Brown.
Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.
J. Physiol. (Lond.)
364:
381-393,
1985[Abstract].
6.
Brown, J. E.,
and
J. R. Blinks.
Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors.
J. Gen. Physiol.
64:
643-665,
1974
7.
Brown, J. E.,
P. K. Brown,
and
L. H. Pinto.
Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III.
J. Physiol. (Lond.)
267:
299-320,
1977[Medline].
8.
Brown, J. E.,
L. B. Cohen,
P. De Weer,
L. H. Pinto,
W. N. Ross,
and
B. M. Salzberg.
Rapid changes of intracellular free calcium concentration.
Biophys. J.
15:
1155-1160,
1975[Medline].
9.
Brown, J. E.,
and
J. E. Lisman.
Intracellular Ca modulates sensitivity and time scale in Limulus ventral photoreceptors.
Nature
258:
252-254,
1975[Medline].
10.
Cervetto, L.,
L. Lagnado,
R. J. Perry,
D. W. Robinson,
and
P. A. McNaughton.
Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients.
Nature
337:
740-743,
1989[Medline].
11.
Cohen, A. I.
An ultrastructural analysis of the photoreceptors of the squid and their synaptic connections. I. Photoreceptive and non-synaptic regions of the retina.
J. Comp. Neurol.
147:
351-361,
1973[Medline].
12.
Condrescu, M.,
H. Rojas,
A. Gerardi,
R. DiPolo,
and
L. Beaugé.
In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange.
Biochim. Biophys. Acta
1024:
198-202,
1990[Medline].
13.
Crespo, L. M.,
C. J. Grantham,
and
M. B. Cannell.
Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes.
Nature
345:
618-621,
1990[Medline].
14.
Hardie, R. C.
Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors.
J. Neurosci.
15:
889-902,
1995[Abstract].
15.
Harkins, A. B.,
N. Kurebayashi,
and
S. M. Baylor.
Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.
Biophys. J.
65:
865-881,
1993[Abstract].
16.
Huppertz, B.,
and
P. J. Bauer.
Na+-Ca2+,K+ exchange in bovine retinal rod outer segments: quantitative characterization of normal and reversed mode.
Biochim. Biophys. Acta
1189:
119-126,
1994[Medline].
17.
Kaplan, R. S.,
and
P. L. Pedersen.
Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B.
Anal. Biochem.
150:
97-104,
1985[Medline].
18.
Kito, Y.,
T. Seki,
and
M. F. Hagins.
Isolation and purification of squid rhabdoms.
Methods Enzymol.
81:
43-48,
1982[Medline].
19.
Lederer, W. J.,
S. He,
S. Luo,
W. duBell,
P. Kofuji,
R. Kieval,
C. F. Neubauer,
A. Ruknudin,
H. Cheng,
M. B. Cannell,
T. B. Rogers,
and
D. H. Schulze.
The molecular biology of the Na-Ca exchanger and its functional roles in heart, smooth muscle cells, neurons, glia, lymphocytes, and nonexcitable cells.
Ann. NY Acad. Sci.
779:
7-17,
1996[Medline].
20.
Levy, S.,
and
A. Fein.
Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors.
J. Gen. Physiol.
85:
805-841,
1985[Abstract].
21.
Lisman, J. E.,
and
J. E. Brown.
The effects of intracellular iontophoretic injection of calcium and sodium ions on the light response of Limulus ventral photoreceptors.
J. Gen. Physiol.
59:
701-719,
1972
22.
Minke, B.,
and
E. Armon.
Activation of electrogenic Na-Ca exchange by light in fly photoreceptors.
Vision Res.
24:
109-115,
1984[Medline].
23.
Minke, B.,
and
M. Tsacopoulos.
Light induced sodium dependent accumulation of calcium and potassium in the extracellular space of bee retina.
Vision Res.
26:
679-690,
1986[Medline].
24.
Minta, A.,
J. P. Y. Kao,
and
R. Y. Tsien.
Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.
J. Biol. Chem.
264:
8171-8178,
1989
25.
Monteith, G. R.,
and
B. D. Roufogalis.
The plasma membrane calcium pumpa physiological perspective on its regulation.
Cell Calcium
18:
459-470,
1995[Medline].
26.
Nicoll, D. A.,
B. R. Barrios,
and
K. D. Philipson.
Na+-Ca2+ exchangers from rod outer segments and cardiac sarcolemma: comparison of properties.
Am. J. Physiol.
260 (Cell Physiol. 29):
C1212-C1216,
1991
27.
Nicoll, D. A.,
S. Longoni,
and
K. D. Philipson.
Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger.
Science
250:
562-565,
1990[Medline].
28.
O'Day, P. M.,
and
M. P. Gray-Keller.
Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors.
J. Gen. Physiol.
93:
473-492,
1989[Abstract].
29.
O'Day, P. M.,
M. P. Gray-Keller,
and
M. Lonergan.
Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors.
J. Gen. Physiol.
97:
369-391,
1991[Abstract].
30.
Payne, R.,
D. W. Corson,
and
A. Fein.
Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors.
J. Gen. Physiol.
88:
107-126,
1986[Abstract].
31.
Perry, R. J.,
and
P. A. McNaughton.
The mechanism of ion transport by the Na+-Ca2+,K+ exchange in rods isolated from the salamander retina.
J. Physiol. (Lond.)
466:
443-480,
1993[Abstract].
32.
Philipson, K. D.,
and
A. Y. Nishimoto.
Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment.
Am. J. Physiol.
243 (Cell Physiol. 12):
C191-C195,
1982[Abstract].
33.
Reeves, J. P.,
and
C. C. Hale.
The stoichiometry of the cardiac sodium-calcium exchange system.
J. Biol. Chem.
259:
7733-7739,
1984
34.
Reiländer, H.,
A. Achilles,
U. Friedel,
G. Maul,
F. Lottspeich,
and
N. J. Cook.
Primary structure and functional expression of the Na/Ca,K-exchanger from bovine rod photoreceptors.
EMBO J.
11:
1689-1695,
1992[Abstract].
35.
Saibil, H. R.
Structure and function of the squid eye.
In: Squid as Experimental Animals, edited by D. L. Gilbert,
W. J. Adelman, Jr.,
and J. M. Arnold. New York: Plenum, 1990, p. 371-397.
36.
Schnetkamp, P. P. M.,
D. K. Basu,
and
R. T. Szerencsei.
Na+-Ca2+ exchange in bovine rod outer segments requires and transports K+.
Am. J. Physiol.
257 (Cell Physiol. 26):
C153-C157,
1989
37.
Tsoi, M.,
K.-H. Rhee,
D. Bungard,
X.-F. Li,
S.-L. Lee,
R. N. Auer,
and
J. Lytton.
Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain.
J. Biol. Chem.
273:
4155-4162,
1998
38.
Ukhanov, K.,
and
R. Payne.
Rapid coupling of calcium release to depolarization in Limulus polyphemus ventral photoreceptors as revealed by microphotolysis and confocal microscopy.
J. Neurosci.
17:
1701-1709,
1997