Inositol polyphosphates regulate zebrafish left-right asymmetry

Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.     

Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits

We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.     

The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase

We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P. We first confirmed that interactions between the cytoplasmic domains of alpha2 that modulate conformational shifts are fundamentally similar to those of alpha1, suggesting that the predilection of alpha2 for E(1) state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha1/alpha2 chimeras indicates that the difference in E(1)/E(2) poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha1/alpha2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E(1) shift of alpha2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E(2)(K(+)) --> E(1) + K(+)), rather than phosphoenzyme (E(1)P --> E(2)P).     

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.     

Na,K-ATPase is essential for embryonic heart development in the zebrafish

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.     

The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.