Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.

To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture electron microscopy after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 A. Also, freeze-fracture electron microscopy demonstrated uniform vesicles with diameters in the range of 700-1,100 A and an average value close to 900 A. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 A and similar to our previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles per vesicle. The data therefore show that active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close fo 90 A in the freeze-fracture micrographs.

Ultrastructure of the sodium pump. Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+,K+)-ATPase.

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 mum. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per mum(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per mum(2) and 390 +/- 170 per mum(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.

Electron microscopic autoradiography of rabbit reticulocytes active and inactive in protein synthesis.

Electron microscopic autoradiography indicates that only a fraction of ribosome-containing reticulocytes (from phenylhydrazine-treated rabbits) is capable of active protein synthesis in vitro. Ribosomes, and, indeed, polyribosomes, do not appear to limit the rate of protein synthesis.

Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase.

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals.

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.

Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules.

The subcellular distribution and segmental variations in location of gp330, a scavenger receptor for filtered proteins in renal proximal tubules, was analyzed. Kidney tissue from rats (4 different strains), rabbits and humans were analyzed by light- and electron microscope immunocytochemistry, using cryosections or Lowicryl sections from cryosubstituted tissue. Gp330 was located mainly in apical coated pits, small and large endocytic vacuoles and in dense apical tubules in the proximal tubule cells. The labeling density was markedly higher in segments 1 and 2 as compared to segment 3 of the proximal tubule. In addition to the location in the early part of the endocytic pathway, gp330 was also present in lysosomes, especially in segments 1 and 2. The lysosomal labeling was not restricted to the membrane, but was also seen in the matrix. Localization of gp330 in lysosomes was confirmed on sections from purified lysosomal fractions from rat renal cortex. The brush border localization of gp330 in proximal tubules exhibited a characteristic segmental variation. In the initial part of segment 1, there was virtually no brush border labeling. In the remaining part of segment 1 and in segment 2, there was a distinct but sometimes patchy labeling of the brush border. In segment 3, groups of microvilli of approximately 10 as seen in sections were intensively labeled from bottom to tip and there were often more than one of these groups on a single cell, the remaining microvilli were unlabeled. No differences in the cellular and subcellular localization of gp330 were observed between species or rat strains. In conclusion, the present study demonstrates that in addition to its location in the early endocytic and recycling pathway, gp330 is also present in microvilli and the protein and degradation products thereof is present in lysosomes, consistent with its role as a protein scavenger receptor.

Ultrastructure of membrane-bound Na, K-ATPase after extensive tryptic digestion.

Membrane-bound Na, K-ATPase was digested with trypsin in the presence of Rb+ to form the stable 19-kDa and smaller fragments of the alpha-chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the alpha-subunit identified in thin sections. However, intramembrane particles defined by freeze-fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis.

Epitope topology of Na,K-ATPase alpha subunit analyzed in basolateral cell membranes of rat kidney tubules.

For topological analysis of integral membrane protein in situ, we used a novel immunoelectron microscopic technique, SDS-digested freeze-fracture replica labeling (SDS-FRL), and oligopeptide-specific antibodies to clarify the sidedness of Na,K-ATPase alpha subunit epitopes in basolateral cell membranes of kidney tubules. Unfixed tissue slices from rat kidney outer medulla were frozen with liquid helium, freeze-fractured, and replicated. After digestion with SDS to solubilize unfractured membranes and cytoplasm, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for immunocytochemistry. Immunogold labeling using antibodies against the N-terminus (Gly1-His13), Leu815-Gln828 and the C-terminus (Ile1002-Tyr1006) was superimposed on the images of the electron microscope protoplasmic fracture face of the basolateral plasma membranes, thus demonstrating cytoplasmic locations of these epitopes. On the contrary, SDS-FRL showed specific binding of Asn889-Gln903 to cross-fractured basolateral plasma membranes suggesting that this epitope is located on the extracellular side of the membrane.

Three-dimensional structure of renal Na,K-ATPase determined by electron microscopy of membrane crystals.

The three-dimensional structure of Na,K-ATPase was determined by electron microscopy and image processing. Tilt series of negatively stained membrane crystals were recorded. The projections were analyzed by Fourier methods and the data combined to a 3-D model. The unit cell contains two rod-shaped stain-deficient regions interpreted as alpha beta-promoters of Na,K-ATPase. The rods are related by dyad axes oriented perpendicular to the membrane. Outside the lipid bilayer the rods contact different protein units on the two sides of the membrane.

Coexistence of different forms of Na,K-ATPase in two-dimensional membrane crystals.

Two-dimensional membrane crystals of renal Na,K-ATPase were analyzed by electron microscopy and image processing. The particular property of the crystals in this work was that they showed unit cell parameters similar to the previously studied p21 crystals but lacked the dyad axis as observed in nominal 0 degrees-projections. A three-dimensional reconstruction revealed that structural differences between alpha beta-units of the enzyme gave rise to the asymmetry. A high degree of two-fold rotational symmetry was observed in the middle of the structure while the protein units had different three-dimensional shapes at levels above and below the central sections. The simultaneous coexistence of different forms of Na,K-ATPase suggests that the conformational flexibility of the enzyme plays an important role in the pumping process.

Topology of Na,K-ATPase alpha subunit epitopes analyzed with oligopeptide-specific antibodies and double-labeling immunoelectron microscopy.

Using four oligopeptide-specific polyclonal antibodies, we mapped the alpha subunit of Na,K-ATPase by double-labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N-terminus (Gly1-His13), C-terminus (Ile1002-Tyr1016) and Leu815-Gln828 are located on the same face of crystallized Na,K-ATPase membranes from pig kidney, whereas the epitope Asn889-Gln903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C-terminus and that Leu815-Gln828 is exposed on the cytoplasmic and Asn889-Gln903 on the extracellular side. The results are consistent with an eight- or ten-segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu815-Gln828 and Asn889-Gln903.

Ultrastructure of the Na, K-ion pump.

Na, K-ATPase has been analysed by electron microscopy to obtain information about the structure of the enzyme and its organization within the membrane. Following negative staining the membrane-bound enzyme was observed as surface particles which on the basis of their size and frequency and the enzymatic and chemical composition of the membranes are interpreted as protomers (alpha beta-units). Freeze-fracture electron microscopy revealed the enzyme as intramembrane particles. Quantitative electron microscope studies suggested that the intramembrane particles are oligomers of the protein unit that forms the surface particles. Following reconstitution of the enzyme into phospholipid vesicles it was demonstrated that similar intramembrane particles represent a protein unit which transports sodium and potassium. Vanadate and magnesium induced the formation of two-dimensional crystals in the membrane fragments of the purified Na, K-ATPase. Further information regarding the shape and dimensions of the protomer was obtained through analysis of electron micrographs of negatively stained crystals with optical diffraction and image reconstruction methods.