Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues

The functional roles of Asp804 and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase alpha subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala808 --> Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp804 and Asp808 were replaced by Ala, Asn, and Glu in the sheep alpha1 subunit and expressed in a mouse cell line where [3H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E1Na, E1ATP, and E2P conformations. K+ competition of ouabain binding to sheep alpha1 and Asp808 --> Glu enzymes displayed IC50 values of 4.11 mM (nHill = 1.4) and 23.8 mM (nHill = 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na+ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill 相似文献   

In situ analysis of Na, K-ATPase alpha1- and alpha3-isoform mRNAs in aging rat hippocampus

Age-related changes in the expression of Na, K-ATPase alpha1- and alpha3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) alpha1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas alpha3 content shows the opposite ratio and (b) alpha3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1-3. In the 24-month-old rats as compared with the young animals, the alpha1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in the perikaryal layers. The alpha3 signal is reduced about threefold (p<0.0001, ANOVA, n=6) in perikaryal layers, is almost completely absent over interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase a isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age related alterations in hippocampal function, before cell loss.     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Na,K-ATPase subunit beta1 knock-in prevents lethality of beta2 deficiency in mice

The beta2 subunit of the Na,K-ATPase displays functional properties of both an integral constituent of an ion pump and an adhesion and neurite outgrowth-promoting molecule in vitro. To investigate whether the beta1 subunit of the Na,K-ATPase can functionally substitute for the beta2 isoform in vivo, we have generated beta2/beta1 knock-in mice by homologous recombination in embryonic stem cells. In beta2/beta1 knock-in mice, expression of beta2 was abolished, whereas beta1 mRNA expression from the mutated gene amounted to approximately 15% of the normal expression of beta2 in the adult mouse brain and prevented the juvenile lethality observed for beta2 null mutant mice. In contrast to beta2 null mutant mice, the overall morphological structure of all analyzed brain regions was normal. By immunohistochemical analysis, beta1 expression was detected in photoreceptor cells in the retina of knock-in mice at an age when expression of beta1 and beta2, respectively, is downregulated and persisting in the wild-type mice. Morphological analysis by light and electron microscopy revealed a progressive degeneration of photoreceptor cells. Apoptotic death of photoreceptor cells determined quantitatively by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis increased in beta2/beta1 knock-in mice with age. These observations suggest that the beta1 subunit of the Na,K-ATPase can substitute sufficiently, at least in certain cell types, for the role of the beta2 subunit as a component of a functional Na,K-ATPase, but they do not allow us to determine the possible role of the beta2 subunit as an adhesion molecule in vivo.     

Differential expression of Na,K-ATPase alpha-isoform mRNAs in aging rat cerebellum

Age-dependent changes in the expression of Na,K-ATPase alpha 1- and alpha 3-mRNAs were analyzed in the rat cerebellum by in situ hybridization. In young rats, alpha 1-mRNA showed prominent labeling in the granular layer (GL) with moderate fine distribution in the molecular layer (ML), Purkinje cell layer (PCL), and white matter (WM) but no clusters over Purkinje cells (PCs). In old rats, alpha 1-mRNA remained unchanged in ML and PCL, but declined by 43% (P < 0.0001) in GL and increased by 624% (P < 0.0001) in WM. alpha 3-mRNA in young rats showed large clusters of label on stellate, basket, Golgi, and PCs and fine grains diffusely in ML, GL, and WM. In old rats, alpha 3-mRNA declined by 87% in ML, 83% in PCL, 84% per PC, and 89% in GL and increased by 111% in WM (all values P < 0.0001) relative to young rats. PC numbers were reduced by 30%, but the average area of PC profiles did not change significantly. In old rats, the specific cluster-like label related to alpha 3-mRNA on PCs, stellate, basket, and Golgi cells was lost. Immunocytochemistry of cerebellum and hippocampus showed no age-related change in the distribution and density of total catalytic polypeptide. Thus, the discordance between changes in the levels of mRNAs in neuronal layers and WM in the face of constant polypeptide levels indicates age-related changes in polypeptide turnover. Cell- and isoform-specificity of alpha-isoform mRNAs in aging rat cerebellum may reflect differential regulation underlying age-related impairments in signal transduction and motor learning.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

Plasticity of Na,K-ATPase isoform expression in cultures of flat astrocytes: species differences in gene expression

The sella and parasellar region may be affected by a variety of disease states. Diseases of this region often result in visual disturbances because of the proximity of the sella to the optic pathways and cranial nerves. Knowledge of the pathological conditions affecting the sella and surrounding structures is important for the orbital imager.     

Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5'-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, beta-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus-tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5'-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination.     

Expression of the rat arginine vasopressin gene in transgenic mice

A line of mice has been developed which are transgenic for an 8.2-kilobase (kb) genomic clone of the rat vasopressin (VP) gene. Using a polymerase chain reaction technique, the rat VP (rVP) transgene was shown to have tissue-specific mRNA expression in the hypothalamus, temporal lobe, parietal cerebral cortex, cerebellum, and posterior pituitary, similar to the tissue distribution of endogenous mouse and rat VP expression. Expression of transgenic rVP mRNA was also found in the lung and pancreas of the transgenic mice, sites of known ectopic expression of VP. Using two methods, Northern blot analysis with species-specific cRNA probes and a quantitative polymerase chain reaction technique, the quantity of rVP transgene mRNA was shown to regulate appropriately in response to an osmotic stimulus. After 72 h of water deprivation, the quantity of transgenic rVP mRNA increased 6.8 +/- 3.0-fold. This was not significantly different than the fold increase in mouse VP mRNA quantity seen in nontransgenic mice (4.8 +/- 1.5) but was significantly different (P < 0.05) than the 1.2 +/- 0.03-fold increase in rat VP mRNA seen in normal rats after water deprivation. In the rat hypothalamus, VP mRNA poly(A) tail length increases with osmotic stimulation, while in the mouse it does not. The poly(A) tail of transgenic rVP mRNA expressed in mouse hypothalamus did not increase in length after osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.