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Complementary DNA encoding the Na/Ca,K‐exchanger was isolated from bovine retina cDNA libraries. The complete full‐length cDNA is approximately 4 kb long and contains an open reading frame of 3597 bp. The deduced amino acid sequence corresponds to a protein of 1199 amino acids with a calculated molecular weight of approximately 130 kDa. Hydrophobicity analysis revealed the presence of two alternating sets of hydrophobic and hydrophilic domains. There also exists a hydrophobic region at the N‐terminus which may be part of a cleavable signal peptide. The protein shares limited sequence homology with the Na/Ca‐exchanger from cardiac sarcolemma. Northern blot analysis indicates that the approximately 6 kb transcript is highly specific for retinal tissue. Insect cells infected with recombinant baculovirus bearing the full‐length cDNA express a functional Na/Ca,K‐exchanger with an apparent relative molecular weight of approximately 210 kDa, as determined by Western blotting.

The cyclic GMP-dependent cation channel from bovine rod outer segments has been purified to >90% homogeneity by a rapid two-step chromatographic procedure. The purified channel has an apparent molecular mass of63 kDa as determined by NaDodSO4/gel electrophoresis. When incorporated into the membrane of liposomes, the purified protein mediates the cyclic GMP-dependent efflux of entrapped Ca2+. The reconstituted channel protein exhibits properties similar to the cyclic GMP-dependent channel observed in excised patches of the plasma membrane and in disk membranes. Cyclic GMP activated the channel cooperatively (Hill coefficient n = 3.1) with an apparent Michaelis constant of -11 ,AM. After reconstitution of the purified protein into a planar lipid bilayer, we recorded cyclic GMP-stimulated single-channel activity. The single-channel conductance at physiological salt concentrations and in the absence of divalent cations was 26 pS. The drug l-cis-diltiazem, shown to block the cyclic GMP-dependent channel in excised patches of the plasma membrane and in isolated disks of rod outer segments, was ineffective against the purified channel.Vertebrate photoreceptors hyperpolarize in response to light because of the closure of cation-specific channels present in the plasma membrane of the outer segment. The coupling of light to channel gating is likely to be mediated by an internal messenger(s) (1). It has recently been demonstrated in rod photoreceptor cells that cyclic GMP, acting on the intracellular side, can open channels in the plasma membrane by a cooperative binding mechanism (2-7) and that divalent cations can block the cyclic GMP-activated channel (5, 6). Cyclic GMP also directly activates a cation conductance in the disk membrane of rod outer segments (8-10) by a cooperative mechanism remarkably similar to that of the plasma membrane channel (11). As a first step towards the biochemical characterization of the cyclic GMP-dependent channel, we have solubilized the channel protein from whole outer segments and functionally reconstituted it into the membrane of liposomes (12). We now report the purification of a 63-kDa protein that, when incorporated into liposome membranes or a planar lipid bilayer, mediates the cyclic GMP-stimulated flux of mono-and divalent cations. METHODSPurification of the Cyclic GMP-Dependent Cation Channel. Rod outer segments (40 mg of rhodopsin) prepared from dark-adapted bovine retinae by a sucrose gradient method (13) were stripped of peripheral proteins by hypotonic extract in HD buffer (10 mM Hepes adjusted with KOH to pH 7.4 and 1 mM dithiothreitol) containing 1 mM EDTA followed by centrifugation at 150,000 x g for 30 min, a procedure repeated three times. The membrane pellet was then dissolved in 30 ml of HD buffer containing 10 mM CaCl2, 18 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% soybean phosphatidylcholine (type IV-S from Sigma), 0.15 M KCl, and the following protease inhibitors: diisopropyl fluorophosphate (0.1 mM), aprotinin (5 ,ug/ml), leu...

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.