Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium adenosinetriphosphatase of bovine retinal rod outer segment disks.

A Ca(2+)-pumping ATPase activity is present in bovine retinal rod outer segment purified disks. The ATPase has a high Ca2+ affinity (KM = 25 microM). Low Ca2+ (n-microM) concentrations stimulate an ATP-dependent Ca2+ uptake and the ATP hydrolysis in the absence of exogenous Mg2+. Electrophoretic analysis of disk proteins after treatment with (gamma-32P)ATP shows the existence of the enzyme-phosphate acid-stable, hydroxylamine-sensitive intermediate complex of molecular mass of about 135 kDa. The results would indicate the presence of an inwardly directed Ca(2+)-ATPase pump acting on the disk membrane, that could be involved in the regulation of cytosolic free Ca2+ levels inside ROS.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

ATP synthesis in rod outer segments of bovine retina by the reversal of the disk Ca(2+) pump

Purified disk membranes from rod outer segments of the bovine retina were able to synthesize ATP with a maximal activity (about 52 nmoles ATP/min/mg of protein) at physiological calcium concentrations. This activity was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting the reversal functioning of the disk Ca(2+)-ATPase, which would act as a ATP synthesizer at the expense of the calcium gradient between the disks and the cytoplasm of the rod outer segment. The results are discussed in terms of the need of an immediate source of ATP on the disk membranes where the energy is required to supply the rapid reactions of the photoreception processes.     

Effect of phorbol esters and diacylglycerols on calcium transport by human platelet membranes

The effects of phorbol esters and diacylglycerols on Ca2+ transport in isolated human platelet membranes were determined. Phorbol 12-myristate 13-acetate (PMA) stimulated Ca2+-ATPase activity in crude and purified internal platelet membranes approximately 2-fold with half-maximal stimulation occurring at 10 nM. Dilauroylglycerol also stimulated Ca2+-ATPase activity half-maximally at a concentration of 7.5 microM, but dioctanoylglycerol was without effect at up to 30 microM. PMA also inhibited Ca2+ uptake when added before or after commencement of ATP-dependent transport. PMA (25 nM) doubled the rate of Ca2+ efflux from passively loaded membranes in the absence of ATP. No protein kinase C activity was detected in crude or purified membranes by histone phosphorylation or endogenous protein phosphorylation assays. These results suggest that PMA and dilauroylglycerol stimulate Ca2+-ATPase activity and inhibit ATP-dependent Ca2+ transport by increasing the permeability of the membranes to Ca2+.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

Na+- and cGMP-induced Ca2+ fluxes in frog rod photoreceptors

We have examined the Ca2+ content and pathways of Ca2+ transport in frog rod outer segments using the Ca2+-indicating dye arsenazo III. The experiments employed suspensions of outer segments of truncated, but physiologically functional, frog rods (OS-IS), intact isolated outer segments (intact OS), and leaky outer segments (leaky OS with a plasma membrane leaky to small solutes, but with sealed disk membranes). We observed the following. Intact OS or OS-IS isolated and purified in Percoll-Ringer's solution contained an average of 2.2 mM total Ca2+, while leaky OS contained 2.0 mM total Ca2+. This suggests that most of the Ca2+ in OS-IS is contained inside OS disks. Phosphodiesterase inhibitors increased the Ca2+ content to approximately 4.2 mM in intact OS or OS-IS, whereas the Ca2+ content of leaky OS was not altered. Na-Ca exchange was the dominant pathway for Ca2+ efflux in both intact and leaky OS/OS-IS. The rate of Na-Ca exchange in intact OS/OS-IS was half-maximal between 30 and 50 mM Na+; at 50 mM Na+, this amounted to 5.8 X 10(7) Ca2+/OS X s or 0.05 mM total Ca2+/s. This is much larger than the Ca2+ component of the dark current. Other alkali cations could not replace Na+ in Na-Ca exchange in either OS-IS or leaky OS. They inhibited the rate of Na-Ca exchange (K greater than or equal to Rb greater than Cs greater than or equal to Li greater than TMA) and, as the inhibition became greater, a delay developed in the onset of Na-Ca exchange. The inhibition of Na-Ca exchange by alkali cations correlates with the prolonged duration of the photoresponse induced by these cations (Hodgkin, A. L., P. A. McNaughton, and B. J. Nunn. 1985. Journal of Physiology. 358:447-468). In addition to Na-Ca exchange, disk membranes in leaky OS showed a second pathway of Ca2+ transport activated by cyclic GMP (cGMP). The cGMP-activated pathway required the presence of alkali cations and had a maximal rate of 9.7 X 10(6) Ca2+/OS X s. cGMP caused the release of only 30% of the total Ca2+ from leaky OS. The rate of Na-Ca exchange in leaky OS amounted to 1.9 X 10(7) Ca2+/OS X s.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Characterization of Ca2+ transport and enzyme activity in microsomes isolated from guinea-pig stomach smooth muscle

Ca2+ uptake by microsomes prepared from guinea-pig stomach required the presence of both ATP and Mg2+ and was unaffected by NaN3. ATP-dependent Ca2+ uptake increased with increasing free Ca2+ concentration from 0.1 to 5 microM, and further increase in Ca2+ concentration above 5 microM did not enhance the uptake further. Half-saturation occurred at approximately 0.55 microM. The t1/2 values of Ca2+ loss from these vesicles loaded in the presence of oxalate were significantly slower than those in the absence of oxalate. Enzyme activity suggested linkage between Ca2+ uptake and ATPase activity, and most of the azide-sensitive component of ATP hydrolysis was attributable to potent inhibition of ADPase activity.     

Molecular mechanisms of photoreception. IV. Ca2+-inhibited GTPase of rod outer segments of the frog retina

Ca2+-dependent GTPase activity is found to be present in the rod outer segments of frog retina. GTPase localization in rod outer segments is shown by fractionating the rod outer segment preparation in the sucrose density gradient. The enzyme is readily washed out of cells with isotonic NaCl solution. The Km is 0.6 mM for GTP. The activity is inhibited by 78 +/- 12% with the increase in Ca2+ concentration from 10(-9) to 10(-7) M. GTP hydrolysis is inhibited by the same concentrations of Ca2+ which block the sodium conductivity of the rod outer segment cytoplasmic membrane.     

Subcellular distribution of ATP-dependent calcium transport in rat duodenal epithelium

Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome -c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker (Na+-K+)-ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter-current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of (Na+-K+)-ATPase, contained ATP-dependent Ca2+-transport activity with Vmax = 3.7 nmol/mg per min and Km = 0.5 microM. A major Golgi-derived population exhibited Ca2+-transport activity with Vmax and Km values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca2+-transport activity with Vmax = 4 nmol/mg per min and Km = 0.5 microM. In addition to demonstrating that ATP-dependent Ca2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca2+-pumping ability.     

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.     

Calcium release from rod outer segments: evidence for a cGMP-sensitive calcium binding protein.

Numerous studies investigating the cGMP-gated cation conductance in rod disk membranes have purported to measure efflux of Ca2+ entrapped in rod disk membrane vesicles. We have utilized sonication and osmotic shock as additional tests for sensitivity of cGMP- and A23187-induced Ca2+ release to elimination of the transvesicular Ca2+ gradient. We find that 1) Treatment with sonication or osmotic shock in low Ca2+ medium does not release Ca2+ from either native cGMP/Ca2(+)-loaded vesicles or solubilized, reconstituted "Ca2(+)-loaded" vesicles, 2) 70-100% of the cGMP-induced "flux" and 90-100% of the A23187-induced Ca2+ "flux" is insensitive to elimination of the Ca2+ gradient by sonication or osmotic shock in low Ca2+ medium, and 3) total amount of releasable Ca2+ is related to membrane surface area rather than vesicle entrapment volume. We conclude that 1) A23187 disrupts binding of Ca2+ to proteins and phospholipids as well as releasing entrapped Ca2+ and 2) a large fraction of the cGMP-induced release observed in rod disk vesicles is due to release of bound Ca2+.     

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea: II. Partial characterization of Ca2+ transport and glyceollin effects

Calcium uptake was examined in sealed plasma membrane vesicles isolated from the plant pathogenic fungus, Phytophthora megasperma f. sp. glycinea. Calcium uptake was ATP-dependent and by the addition of various ionophores in the presence of ATP, it was shown that Ca2+ transport was mediated by a nH+/Ca2+ antiport. Further evidence for this antiport mechanism included Ca2+ uptake driven by an imposed pH gradient and the observation that calcium could dissipate a steady-state pH gradient across the vesicle membrane. Transport mediated by the nH+/Ca2+ antiport was optimal at pH 7.0, and demonstrated saturation kinetics for Ca2+ with a Km of about 7 microM. Glyceollin, a soybean phytoalexin, was found to inhibit Ca2+ transport consistent with its ability to increase H+ conductance. In the presence of glyceollin, calcium leakage from Phytophthora membrane vesicles also increased. This study provides basic information about calcium transport in a plant pathogenic fungus as well as demonstrating a possible mode of action of a phytoalexin.     

Calcium-hydrogen exchange in isolated bovine rod outer segments

We have measured Ca-H exchange in rod photoreceptors with different preparations of rod outer segments isolated from bovine retinas (ROS). One preparation contained ROS with an intact plasma membrane (intact ROS), and in the other preparation, the plasma membrane was leaky to small solutes (leaky ROS) and the cytoplasmic space was freely accessible to externally applied solutes. Addition of Ca2+ to Ca2+-depleted ROS (both intact and leaky) resulted in uptake of Ca2+ that was accompanied by the release of protons when catalytic amounts of the ionophore A23187 were present. This ionophore mediates Ca-H exchange transport across ROS membranes and serves to gain access to the intracellular compartment where Ca-H exchange appears to take place. Two protons were ejected for each calcium ion taken up. Conversely, when protons were added to Ca2+-enriched ROS, Ca2+ was released in the presence of A23187. The majority of this Ca-H exchange was observed only when A23187 was present in both intact and leaky ROS. We conclude that Ca-H exchange occurs predominantly in the intradiskal space and at the surface of the disk membrane rather than across the disk membrane. These exchange binding sites can accommodate 10 mol of Ca2+/mol of rhodopsin at physiological pH. We were unable to detect any Ca2+ release when a proton gradient was rapidly established across the disk membrane in the absence of A23187. These results are discussed in relation to the hypothesis that protons produced by the light-induced hydrolysis of cGMP cause the release of Ca2+ into the cytoplasm of rod photoreceptor cells.     

Inhibition by orthovanadate of ATP-dependent Ca2+ transport in microsomes isolated from rat liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5'-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at approximately 10 microM vanadate, corresponding to approximately 12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Localization of several rhodopsin regeneration enzymes in retinal rods]

The distribution of NAD kinase and glucose-6-phosphate dehydrogenase within membranes of both outer and inner retina rod segments was studied by the sucrose gradient centrifugation of crude outer segment preparations. Rhodopsin and retinoldehydrogenase served as markers for outer segment membranes, whereas succinate dehydrogenase was a marker for inner ones. It is shown that NAD kinase and glucose-6-phosphate dehydrogenase are localized within inner segment membranes of the photoreception cell and that the activity of these enzymes in the crude preparations is due to contamination of the inner segments.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.