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K Takeyasu

Publications and source records attributed to K Takeyasu.

At least 55 records · Page 3Linked to original sources
PubMed1996

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy.

The structure of Escherichia coli F0F1-ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping-mode operation. The majority of F0F1-ATPases were visualized as spheres with a calculated diameter of approximately 90 angstroms, and a height of approximately 100 angstroms from the membrane surface. F0 sectors were visualized as two different ring-like structures (one with a central mass and the other with a central hollow of greater than or equal to 18 angstroms depth) with a calculated outer diameter of approximately 130 angstroms. The two different images possibly represent the opposite orientations of the complex in the membranes. The ring-like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Cell Membrane↗
PubMed1995

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.

Adenosine Triphosphatases↗
PubMed1994

Assembly of Na,K-ATPase alpha-subunit isoforms with Na,K-ATPase beta-subunit isoforms and H,K-ATPase beta-subunit.

cDNA encoding an epitope tag was joined to cDNAs encoding the chicken Na,K-ATPase beta 1 and beta 2 and H,K-ATPase beta-subunits to allow recognition of these beta-subunits with the same monoclonal antibody during assembly assays. cDNAs encoding chicken Na,K-ATPase alpha 1, alpha 2, or alpha 3 and Na,K-ATPase beta 1 or beta 2 or H,K-ATPase beta-subunits were transiently coexpressed in mammalian cells. Subunit assembly was assayed by immune precipitation of alpha-isoforms with a monoclonal antibody to the epitope-tagged beta-subunits. Each of the chicken alpha-isoforms assembled with each of the Na,K-ATPase beta-subunits and the H,K-ATPase beta-subunit. Each of the epitope-tagged beta-subunits also assembled with a Na,K-ATPase/Ca-ATPase chimera that retained only 26 amino acids of the Na,K-ATPase alpha-subunit, demonstrating that all three beta-subunits recognize this same alpha-subunit assembly site.

Amino Acid Sequence↗
PubMed1994

Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.

Amino Acid Sequence↗
PubMed1994

Molecular imaging of Na+,K(+)-ATPase in purified kidney membranes.

Ion channels and pumps in cell membranes consist of multiple transmembrane segments that are thought to be critical for transport of ions. Channel structures constituted by these transmembrane segments are characteristic of ion channels, whereas such structures have not been identified in ion pumps until now. By applying atomic force microscopy on Na+,K(+)-ATPase molecules in canine kidney membranes under tapping mode, we identified a hollow in the protein with a characteristic internal diameter of 6-20 A and an external diameter of 20-55 A depending upon treatment conditions. This hollow may be interpreted as a channel-like conformation of Na+,K(+)-ATPase. In the regions where the proteins were absent, lipid head structures with 2 A width and 6 A length were imaged in an orthorhombic lattice.

Animals↗
PubMed1994

26 amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are sufficient for assembly with the Na,K-ATPase beta-subunit.

Chimeric cDNAs encoding a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase alpha-subunit were constructed to seek the minimal region of the alpha-subunit sufficient for assembly with the Na,K-ATPase beta-subunit. cDNAs encoding a chimera and the chicken beta-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken beta-subunit. A chimera containing 26 amino acyl residues of the Na,K-ATPase alpha 1-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asn894 to Ala919) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr871 to Thr898) was able to assemble with the chicken beta-subunit. This alpha-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full-length beta-subunit also assembled with a beta-subunit chimera that retained only the ectodomain of the chicken beta 1-subunit. These results suggest that the Na,K-ATPase alpha-subunit has the same topology in the membrane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membrane-spanning domains, and that the aminoacyl residues between membrane domains H7 and H8 are involved in assembly with the beta-subunit in the extracellular/lumenal space.

Amino Acid Sequence↗
PubMed1994

Primary structure of avian H+/K(+)-ATPase beta-subunit.

A cDNA encoding a beta-subunit of the avian H+/K(+)-ATPase was cloned from a chicken stomach cDNA library, and its nucleotide sequence determined. A comparison between all the available sequence data for the beta-subunits of P-type ATPases reveals several evolutionarily conserved regions. Overall identity was 66% when compared with mammalian H+/K(+)-ATPase beta-subunits, 34% identity when compared with the Na+/K(+)-ATPase beta 2-subunits, and 33% identity when compared with the Na+/K(+)-ATPase beta 1-subunits.

Amino Acid Sequence↗
PubMed1994

Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells.

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na+ and K+ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.

Animals↗
PubMed1994

Analysis of subunit assembly of the Na-K-ATPase.

The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.

Amino Acid Sequence↗
PubMed1993

Ca(2+)-dependent and thapsigargin-inhibited phosphorylation of Na+,K(+)-ATPase catalytic domain following chimeric recombination with Ca(2+)-ATPase.

Two chimeric proteins comprising the Na,K-ATPase catalytic domain (large cytosolic loop) and the two flanking regions of the Ca-ATPase were obtained by transient or stable expression in mammalian cells transfected with recombinant DNA. In the first chimera (CpNC), a large portion (containing the nucleotide-binding site) of the cytosolic loop between putative membrane spans M4 and M5 of the sarcoendoplasmic reticulum Ca2+ (SERCA) 1 (fast muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. In the second chimera (CNpC), an even larger portion (containing the nucleotide-binding site and the phosphorylation site) of the analogous cytosolic loop of the SERCA2 (cardiac muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. Steady state Ca2+ transport and coupled ATP hydrolysis by the chimeric proteins were negligible as compared to those obtained with SERCA enzymes. Nevertheless, the chimeric proteins were able to utilize ATP to form phosphoenzyme levels equal to those formed by SERCA ATPases. Chimeric and SERCA enzymes exhibited an identical Ca2+ requirement for ATP utilization and sensitivity to thapsigargin (TG) which is a specific inhibitor of SERCA ATPase and not of Na,K-ATPase. Furthermore, both SERCA and chimeric enzymes could be phosphorylated with P(i), and this reaction required removal of Ca2+. In comparative experiments, the functional pattern of seemingly unaffected phosphoenzyme formation and inhibited Ca2+ transport was produced in the SERCA ATPase even by single mutation of Pro337 to Ala, evidently due to defective protein conformation. Retention of Ca2+ and TG sensitivity by the chimeric proteins demonstrates that the Ca(2+)- and TG-binding domains do not reside within the cytosolic loop replaced by chimeric substitution and strongly support previous studies suggesting that binding of calcium required for enzyme activation occurs within the membrane-bound region of the SERCA ATPases (Clarke et al., 1989a; Sumbilla et al., 1991).

Amino Acid Sequence↗
PubMed1993

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.

Amino Acid Sequence↗
PubMed1993

Structural analysis and expression of a chromosomal gene encoding an avian Na+/K(+)-ATPase beta 1-subunit.

Chicken chromosomal DNA encoding the Na+/K(+)-ATPase beta 1-subunit was cloned and characterized. Its exon-intron structure is identical to mammalian (human and rat) beta 1-subunit genes. The transcription initiation site, TATA box, and an ATTGG (antisense CCAAT) sequence follow approximately 1 kilobase of GC-rich 5' upstream sequence that contains many consensus sequences for transcription factors whose relative positions are conserved between human and chicken genes. When this beta 1-subunit gene was stably incorporated into mouse L cells and C2C12 cells, the avian beta 1-subunit was expressed under the control of the its own promoter.

Amino Acid Sequence↗
PubMed1993

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

Amino Acid Sequence↗
PubMed1993

Expression of dopamine D1A receptor gene in proximal tubule of rat kidneys.

The existence of dopamine receptor subtypes outside the central nervous system has been suggested by pharmacological and biochemical techniques. Whether the renal dopamine receptors are distinct from those cloned from the brain remains to be determined. Indeed, the expression of any of these receptor genes in the kidney has not been demonstrated definitively. In this study, we amplified D1A receptor cDNA from microdissected proximal convoluted tubules of the rat kidney by reverse transcription-polymerase chain reaction; primers were based on the sequence of rat D1A cDNA cloned from the brain and corresponded to the third cytoplasmic loop of the receptor. Specificity of the amplified products was verified by restriction analysis, Southern blots, and sequencing. Furthermore, solution hybridization indicated the presence of a single ribonuclease-protected RNA species corresponding to the D1A receptor mRNA in proximal tubules. These studies report for the first time the presence of D1A receptor message in the kidney.

Animals↗
PubMed1993

Polarized distribution of Na,K-ATPase in honeybee photoreceptors is maintained by interaction with glial cells.

Arthropod photoreceptors are polarized cells displaying distinct surface domains. The distribution of the Na,K-ATPase (sodium pump) over these domains was examined in the honeybee photoreceptor using a monoclonal antibody that specifically recognizes the sodium pump alpha-subunit (approximately 100 kDa). We find that the sodium pump is restricted to sites of the nonreceptive photoreceptor surface closely juxtaposed to glial cells; no sodium pumps were detected on the glia-free domains of the nonreceptive surface and on the light-sensitive microvillar membranes. In order to determine the role of photoreceptor-glia contact in maintaining this polarized pump distribution, we assayed the distribution of the Na,K-ATPase after experimentally influencing photoreceptor-glia contact. Sodium pumps were present on the entire nonreceptive photoreceptor surface when photoreceptor-glia contact was removed by isolating the photoreceptors. Remodeling photoreceptor-glia contact by incubation in hyperosmotic saline caused a redistribution of sodium pumps on the photoreceptor surface corresponding to the redistribution of glial cells. We show, further, that both photoreceptor-glia contact and Na,K-ATPase distribution are independent of extracellular Ca2+. No junctional structures were observed at the borders between Na,K-ATPase-positive and Na,K-ATPase-negative membrane domains. Together, these results suggest that adhesion of glial cells to the photoreceptors plays a crucial role in the maintenance of the polarized distribution of Na,K-ATPase in the honeybee photoreceptors. The Ca(2+)-independent adhesion of glial cells to the photoreceptor surface may trap the pump molecules at the sites of photoreceptor-glia contact.

Animals↗
PubMed1993

D1A dopamine receptor stimulation inhibits Na+/K(+)-ATPase activity through protein kinase A.

We stably expressed the rat D1A dopamine receptor in mouse fibroblast LTK- cells and obtained specific ligand binding and functional activity characteristic of the D1A dopamine receptor coupled to stimulation of adenylyl cyclase. In the transfected cells, the selective D1 agonist fenoldopam caused a concentration-dependent inhibition of Na+/K(+)-ATPase activity, achieving maximum inhibition of approximately 30%. The latter was abolished by the selective D1 antagonist (+)-SCH 23390 and by the specific protein kinase A inhibitor protein kinase inhibitor-(6-22) amide. In the nontransfected cells, fenoldopam did not affect Na+/K(+)-ATPase activity. 8-Chlorophenylthio-cAMP inhibited Na+/K(+)-ATPase activity in both transfected and nontransfected cells; this effect was blocked by protein kinase inhibitor-(6-22). These results indicate that the inhibition of Na+/K(+)-ATPase activity induced by agonist occupancy of D1A receptors is mediated by protein kinase A.

Adenylyl Cyclases↗
PubMed1992

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.

Animals↗