Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes

Paul J. Bauer1, Heike Schauf1, Andreas Schwarzer1, and Joel E. Brown2

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


    ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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+ · s-1 · 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


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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).


    RESULTS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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+ · min-1 · 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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Fig. 1.   Na+-induced Ca2+ efflux from vesicles prepared from squid rhabdomeric membranes. Vesicles were suspended in 1 ml of a dye solution that contained 100 mM KCl, 10 mM HEPES (pH 7.4 adjusted with LiOH), 50 µM arsenazo III, and 179 (a), 169 (b), or 178 µg (c) protein. Additions were 50 µl of a 2 M solution of indicated alkali ion in dye solution described above. Ionophore and CaCl2 were added twice in increments of 2.5 µM (A-23187) and 2 µM (CaCl2). The 1st additions of alkali ions were made at time 0; 2nd additions were made after 2 min. Ordinate is absorbance difference (Delta OD) between 652 and 700 nm (dual-wavelength mode). For clarity, recordings were arbitrarily shifted along ordinate. Downward deflections after addition of alkali ions were due to decreases in apparent Ca2+ affinity of metallochromic dye arsenazo III. Downward deflections after ionophore addition were due to Ca2+ complexation by A-23187. Initial passive Ca2+ effluxes (in nmol · min-1 · mg protein-1) were 1.60 (a), 1.50 (b), and 1.45 (c). Temperature, 4°C. OD, optical density.

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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Fig. 2.   Na+ and A-23187 act on a common Ca2+ pool. Consecutive Ca2+ releases elicited by a variable amount of Na+ followed by an excess of ionophore add up to same total amount of releasable Ca2+. Additions (arrowheads) were as follows (final concns): Na+, 10 mM (a) and 95 mM (b); A-23187, 2.5 µM. Protein, 214 µg/ml in each experiment. Downward deflections on addition of Na+ are not shown. Recordings have been rescaled to indicate directly changes in moles of Ca2+ per mg protein, by using Ca2+ calibrations made within each cuvette (as shown in Fig. 1). Passive Ca2+ effluxes before Na+ addition are indicated by dashed lines; initial Ca2+ fluxes, measured as the slope 10 s after Na+ addition, are indicated by dotted lines. Each cuvette contained vesicles prepared by separate extrusions; therefore, values of total Ca2+ contained differed slightly for the 2 cuvettes.

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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Fig. 3.   Ca2+ efflux measured with fluo 3. These measurements, like those of Fig. 1, show Na+-induced Ca2+ efflux from vesicles prepared from rhabdomeric membranes of squid retinas. Ordinate is change in fluorescence (excitation 480 nm, emission >520 nm) of fluo 3 after subtraction of fluorescence just before beginning of trace. Dye solution (at 5°C) was composed of 100 mM KCl, 10 mM HEPES adjusted to pH 7.4 with KOH, and 10 µM fluo 3. The 1st addition was made at time 0. Additions (arrowheads; final concns) for A: 39 mM NaCl, 4 µM ionomycin, 0.005% Triton X-100, 1 µM CaCl2 (twice); for B: 39 mM KCl, 39 mM NaCl. The two curves were normalized for response to addition of Ca2+ to each cuvette. Recordings were obtained with different extrusions of vesicles; therefore, vesicles contained slightly different total concentrations of Ca2+.

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+ · s-1 · 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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Fig. 4.   Titration of the Na+-induced Ca2+ efflux. Na+-induced Ca2+ effluxes were determined 10 s after adding Na+ by subtracting the passive Ca2+ efflux measured before Na+ addition. Data have been normalized by amount of protein present in each cuvette (mean 202 ± 14 µg/ml). Best fit of Hill equation (solid curve) yielded a half-maximum value (K1/2) of 36.4 ± 5.9 mM Na+, a Hill coefficient of 1.64, and a maximal Na+-induced Ca2+efflux of 0.74 nmol Ca2+ · s-1 · mg protein-1. Dotted line: maximal Na+-induced Ca2+ efflux.

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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Fig. 5.   Na+/Ca2+ exchange in presence of various monovalent cations. Ca2+-loaded vesicles were made from squid rhabdomeric membranes that were prepared in absence of K+ by using LiCl instead of KCl in all solutions. Loading buffer contained 10 mM Ca2+, 100 mM indicated alkali ion, and 10 mM HEPES-LiOH, pH 7.4. External Ca2+ was removed with Chelex 100 prepared with corresponding alkali ion. Na+/Ca2+ exchange in presence of different alkali ions (indicated for each curve) is shown. In each experiment, concentration of alkali ion was 100 mM both inside and outside the vesicles. Na+ and A-23187 were added to final concentrations of 95 mM and 2.5 µM, respectively. Dye solution contained 50 µM arsenazo III + 10 mM HEPES adjusted to pH 7.4 with LiOH. Recordings were shifted an arbitrary distance along ordinate and were rescaled to indicate changes in Ca2+ concentration. Dashed lines, linear extrapolations of the passive leakages recorded before addition of Na+; dotted lines, initial Ca2+ fluxes. Protein concentrations of K+, Li+, and Cs+ experiments were 199, 185, and 200 µg/ml, respectively.

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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Fig. 6.   Na+-induced Ca2+ uptake. Rhabdomeric membranes were prepared in absence of K+ by using LiCl instead of KCl in all solutions. For a, vesicles were extruded to include 100 NaCl + 10 mM HEPES (adjusted to pH 7.4 with LiOH); resulting vesicle suspension was diluted 1:5 with 100 mM KCl + 10 mM CaCl2 . For b, vesicles were extruded to include 100 NaCl + 10 mM HEPES (adjusted to pH 7.4 with LiOH); the resulting vesicle suspension was diluted 1:5 with 100 mM LiCl + 10 mM CaCl2. For c, vesicles were extruded to include 100 KCl + 10 mM HEPES (adjusted to pH 7.4 with LiOH); the resulting vesicle suspension was diluted 1:5 with 100 mM KCl + 10 mM CaCl2. In each case, vesicles were incubated for 30 min on ice, Ca2+ not included was removed with Chelex (Li+ form), arsenazo III (in the Li+ form) was added to a concentration of 50 µM, and Ca2+ uptake was probed by adding ionophore A-23187 in an incremental concentration of 2.5 µM (twice). Curves were shifted arbitrarily along ordinate. c (K+, no Na+ inside) shows amount of Ca2+ entrapped because of leakage. Additional entrapment in a and b (Na+, no K+ inside) is due to Ca2+ transported into vesicles by Na+-induced Ca2+ uptake. This Na+/Ca2+ exchange was approximately the same with either K+ or Li+ outside the vesicles.


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Fig. 7.   Forward and reverse Na+/Ca2+ exchange. Vesicles were formed by extrusion of squid rhabdomeric membranes in 100 mM NaCl () or in 100 mM KCl (open circle ). Vesicle suspensions were diluted 1:5 with 100 mM KCl, and CaCl2 was added to a final concentration of 10 mM. Vesicles were incubated for times indicated on abscissa. External Ca2+ was removed by Chelex (K+ form). A: total Ca2+ releasable with 5 µM A-23187. B: Ca2+ efflux evoked on addition of NaCl to a final concentration of 182 mM. Each data point was determined in a different experiment.

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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Fig. 8.   Na+/Ca2+ exchange enhanced by limited proteolysis. A: after extrusion in presence of 10 mM Ca2+, vesicles were incubated in 2 µg/ml trypsin at 4°C for following periods of time (min): 0 (a; control), 1 (b), 2 (c), 4 (d), 6 (e), and 10 (f). Ca2+ effluxes were induced by addition of 95 mM NaCl (final concn) at time 0. Changes in absorbance have been converted to changes in Ca2+ concentration (Delta Ca2+). B: increase of Ca2+ efflux vs. the extent of proteolysis. The single-exponential fit yields a half-maximal stimulation after 2.2 min of proteolysis.

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).


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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