No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate
Abstract
To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate.
Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software.
There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma.
Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.
... To keep cell maintenance, Na + /K + ATPase controls ionic gradients. The ratio between Na + and K + adjusted intracellular pH that regulates absorption and excretion in epithelial cells (Mobasheri et al., 2003). Na + /K + ATPase is a transmembrane protein complex consisted of double isoform combinations of ɑ-catalytic, βregulatory and γ-modulatory subunits working as a key energy driver protecting ionic and osmotic balance in cells. ...
... Prostate gland is responsible for anion citrate synthesis and excretion which depends on accumulation of aspartate and glucose as a part of sufficient role of Na + /K + ATPase transportation system in prostate epithelial cells to keep rapid supply of citrate anion. In the presence of Na + /K + ATPase inhibitors, the accumulation of asparate is fluctuated followed with insufficient citrate production that consequently suppress prostate epithelial cell metabolism and growth (Mobasheri et al., 2003). Moreover, the α-1 subunit is Recognizedby its essential function in cell/cell adhesion as well as cell/matrix interaction. ...
This work was prompted to investigate effect of the vitex berries crude extract on prostate cancer relevant to the cancer growth rate limiting enzymes (5-α reductase and Na⁺/K⁺ ATPase, tumor biomarker; prostate specific antigen (PSA), carcinoembryonic antigen (CEA), sexual hormones; testosterone and luteinizing hormone, anti-inflammatory markers and antioxidant markers. Vitex berries crude extract was prepared and tested against human prostate cancer cell line (PC3) and then incorporated into in-vivo model for prostate cancer induced chemically in rats using chronic administration of testosterone (3mg/ kg/ 90 days) in a sub-cutaneous route and intraperitoneal injection of carcinogenic material, 7,12-Dimethylbenz(a)anthracene (DMBA) at dose of 65 mg/kg as a single dose. Vitex extract at dose of 165 mg/kg/day inhibited cell growth of PC3 which were completely killed at 100μg/ml. In in-vivo study, Vitex extract showed potent anti-prostate cancer evident as significant reduction in cancer growth rate limiting enzymes; 5-α reductase, Na⁺/K⁺ ATPase and prostate specific antigen with suppression in carcinoembryonic antigen production, which amplified by prostate cancer induction. A selective anti-inflammatory effect was recorded with vitex administration represented as significant inhibition in cyclooxygenase-2 and non-significant effect on cyclooxygenase-1. It showed ameliorative effects on sexual hormones showed in amendment in testosterone and luteinizing hormone affected by prostate cancer induction. Investigations of this work prove vitex extract has potential role as anti-prostate cancer evaluated by in-vitro and in-vivo protocols. The anti-prostate cancer properties of vitex extract may be due to its effect against cancer risk factors; antioxidant and anti-inflammatory characters, ameliorative effect on sex hormones and suppression in cancer growth rate limiting enzymes.
... purification α6F, an antibody that is specific for the α1 isoform of the Na + /K + ATPase (Arteaga et al., 2004; Drummond et al., 1998; Hlivko et al., 2006; Mobasheri et al., 2003; Wetzel et al., 1999; Zhang and Ng, 2007) was obtained from a hybridoma cell culture to be used for Western blot and immunohistochemical studies. Briefly, α6F hybridoma cells developed by Dr. D.M. Fambrough were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. ...
... We used a monoclonal antibody (α6F) that is specific for this isoform. This antibody has been widely employed to distinguish Na + /K + ATPase α1 from the other isoforms by immunohistochemistry and Western blot in different tissues, including the retina, of various animal models (Arteaga et al., 2004; Drummond et al., 1998; Hlivko et al., 2006; Mobasheri et al., 2003; Wetzel et al., 1999; Zhang and Ng, 2007). Moreover, the epitope that is recognized by this antibody includes one of the polypeptide sequences that we had identified in our mass spectrometry analysis (highlighted in figure 1A and red in supplementaryfigure 1). ...
Adult newts are able to regenerate their retina and lens after injury or complete removal through transdifferentiation of the pigmented epithelial tissues of the eye. This process needs to be tightly controlled, and several different mechanisms are likely to be recruited for this function. The Na(+)/K(+) ATPase is a transmembrane protein that establishes electrochemical gradients through the transport of Na(+) and K(+) and has been implicated in the modulation of key cellular processes such as cell division, migration and adhesion. Even though it is expressed in all cells, its isoform composition varies with cell type and is tightly controlled during development and regeneration. In the present study we characterize the expression pattern of Na(+)/K(+) ATPase alpha1 in the adult newt eye and during the process of lens and retina regeneration. We show that this isoform is up-regulated in undifferentiated cells during transdifferentiation. Such change in composition could be one of the mechanisms that newt cells utilize to modulate this process.
... The inhibitory effect of 4-AP on the citrate current would suggest that a K + channel may be a part of a regulatory pathway involved in recycling the citrate-coupled K + efflux. In addition, H + -K + -ATPase has been localized to the basal membrane and could pump K + into the cells (Mobasheri et al. 2003). ...
... It is likely that the K + ions transported alongside citrate are regulated by a voltage-gated K + channel (Fig. 3A) and/or other counter-balancing ion transporters (e.g. Na + -K + -ATPase, H + -K + -ATPase) also known to be expressed in prostatic epithelia (Mobasheri et al. 2001(Mobasheri et al. , 2003. ...
Although prostate synthesizes and releases large amounts of citrate, the mechanism of the release is not well understood. Most known citrate transporters mediate uptake of citrate from extracellular space and, consequently, are driven by the transmembrane Na+ gradient, which would not be appropriate for prostatic function. In the present study, we investigated citrate transport in a normal human prostate cell line, PNT2-C2, using mainly electrophysiological methods. Intracellular application of citrate through the patch pipette in the whole-cell recording mode induced an outward current whilst in response to extracellular citrate an inward current was recorded. Membrane currents induced by citrate were bigger than those elicited by other (equimolar) Krebs cycle intermediates. Both inward and outward citrate-induced currents had the same ionic dependence, inhibitor profile and reversal potential. In particular, the currents were strongly dependent on the transmembrane K+ gradient. Uptake and release of citrate and their K+ dependence were confirmed by spectrophotometric enzyme analyses. Citrate-induced membrane currents were also sensitive to pH, consistent with the transporter preferring the trivalent form. Application of intracellular Zn2+ generated an outward current which had the same quantitative K+ dependence as the citrate-induced currents. Extracellular application of a membrane-permeant Zn2+ chelator generated an inward current. These experiments suggested that m-aconitase was tonically active in PNT2-C2 cells. Determination of 'forward' and 'reverse' K+ stoichiometry both suggested a citrate: K+ ratio of 1: 4. We conclude that normal prostatic epithelial cells possess an electrogenic citrate transporter which mediates the cotransfer of 1 trivalent citrate anion alongside 4 K+ out of cells and thus generates a net outward current.
... They should thus promote net Cl − and fluid reabsorption or net Cl − and NH 4 + secretion in tubular and alveolar glands (Wall et al. 1995;Bergeron et al. 2003;Garneau et al. 2017;Marcoux et al. 2017). In certain epithelia such as those of the proximal nephron and gastric glands, they probably play a minor role in these processes given the transported ions are recycled back to the cytosol once they have exited the cell (Boettger et al. 2002;Mobasheri et al. 2003;Fujii et al. 2008Fujii et al. , 2009. In all likelihood, the purpose of K + -Cl − or NH 4 + -Cl − cotransport would then be to sustain the activity of other transport systems by providing them with a constant supply of substrates. ...
In the early 80s, renal microperfusion studies led to the identification of a basolateral K⁺‐Cl⁻ cotransport mechanism in the proximal tubule, thick ascending limb of Henle and collecting duct. More than ten years later, this mechanism was found to be accounted for by three different K⁺‐Cl⁻ cotransporters (KCC1, KCC3 and KCC4) that are differentially distributed along the renal epithelium. Two of these isoforms (KCC1 and KCC3) were also found to be expressed in arterial walls, the myocardium and a variety of neurons. Subsequently, valuable insights have been gained into the molecular and physiological properties of the KCCs in both the mammalian kidney and cardiovascular system. There is now robust evidence indicating that KCC4 sustains distal renal acidification and that KCC3 regulates myogenic tone in resistance vessels. However, progress in understanding the functional significance of these transporters has been slow, probably because each of the KCC isoforms is not identically distributed among species and some of them share common subcellular localizations with other KCC isoforms or sizeable conductive Cl⁻ pathways. In addition, the mechanisms underlying the process of K⁺‐Cl⁻ cotransport are still ill defined. The present review focuses on the knowledge gained regarding the roles and properties of KCCs in renal and cardiovascular tissues.
... Cultured cells not included. Floyd et al., 2010 Shyjan andUrayama et al., 1989;Ghosh et al., 1990;Zlokovic et al., 1993;Sweadner et al., 1994;Lecuona et al., 1996;Arystarkhova and Sweadner, 1997;Mobasheri et al., 1997Mobasheri et al., , 2003Zhang et al., 1997;Wetzel et al., 1999;Blanco et al., 2000;He et al., 2001;Peters et al., 2001;Wetzel and Sweadner, 2001;Cholet et al., 2002;Hoffman et al., 2002;Esplin et al., 2003;Hlivko et al., 2006;McLean et al., 2009;Newton et al., 2009;Floyd et al., 2010;Bottger et al., 2011;Jimenez et al., 2011a;Baker Bechmann et al., 2016;Habeck et al., 2016. genetic diseases linked with mutations in any of the beta subunits. ...
The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.
... We cannot rule out the possibility that some ANO7 is trafficked to the plasma membrane because we have not been able to identify a reliable apical membrane marker. Surprisingly, although the Na ϩ -K ϩ -ATPase has been reported to be apical in some studies (22) but not others (23), in our hands it is clearly basolateral in human prostate. Similarly, H ϩ -K ϩ -ATPase, which has been reported to be apical in mouse (27), is intracellular punctate in human. ...
Ca(2+)-activated Cl(-) channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3-7 in HEK293 cells did not generate Cl(-) currents activated by intracellular Ca(2+), as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease.
Cellular ionic balance relies on ion channels and coupled transporters to maintain and use the transmembrane electrochemical gradients of the cations Na⁺ and K⁺. High intracellular K⁺ concentration provides a ready reserve within the body allowing epithelia to secrete K⁺ into the fluid covering the apical membrane in the service of numerous physiologic activities. A major role for transepithelial K⁺ secretion concerns the balance of total body K⁺ such that excretion of excess K⁺ in the diet safeguards against disturbances to cellular balance. Accomplishing this transepithelial flow involves two archetypical cellular mechanisms, Na⁺ absorption and Cl⁻ secretion. Ion channels for K⁺, Na⁺, and Cl⁻, as well as cotransporters, exchangers, and pumps contribute to produce transepithelial flow by coupling electrochemical gradients such that K⁺ flow enters across the basolateral membrane and exits through the apical membrane. Beyond excretion, transepithelial K⁺ secretion serves to create the high K⁺ concentration of endolymph in the inner ear that supports the sensation of sound and body orientation. For several epithelia such as those in airways and gastric mucosa, the elevated K⁺ concentration of apical fluid may occur largely as a consequence of supporting the secretion of other ions such as Cl⁻ or H⁺. Less well-appreciated consequences of K⁺ secretion may result as in saliva and colonic luminal fluid where a high K⁺ concentration likely influences interactions with the resident microbiome. Independent control of K⁺ secretion also allows for specific adjustments in rate that serve the physiology of organs large and small.
A K(+)-Cl(-) cotransport system was documented for the first time during the mid-seventies in sheep and goat red blood cells. It was then described as a Na(+)-independent and ouabain-insensitive ion carrier that could be stimulated by cell swelling and N-ethylmaleimide (NEM), a thiol-reacting agent. Twenty years later, this system was found to be dispensed by four different isoforms in animal cells. The first one was identified in the expressed sequence tag (EST) database by Gillen et al. based on the assumption that it would be homologous to the Na(+)-dependent K(+)-Cl(-) cotransport system for which the molecular identity had already been uncovered. Not long after, the three other isoforms were once again identified in the EST databank. Among those, KCC4 has generated much interest a few years ago when it was shown to sustain distal renal acidification and hearing development in mouse. As will be seen in this review, many additional roles were ascribed to this isoform, in keeping with its wide distribution in animal species. However, some of them have still not been confirmed through animal models of gene inactivation or overexpression. Along the same line, considerable knowledge has been acquired on the mechanisms by which KCC4 is regulated and the environmental cues to which it is sensitive. Yet, it is inferred to some extent from historical views and extrapolations.
The goal of this study was to define Na,K-ATPase α and β subunit isoform expression and isozyme composition in colorectal cancer cells and liver metastases. The α1, α3 and β1 isoforms were the most highly expressed in tumor cells and metastases; in the plasma membrane of non-neoplastic cells and mainly in a cytoplasmic location in tumor cells. α1β1 and α3β1 isozymes found in tumor and metastatic cells exhibit the highest and lowest Na+ affinity respectively and the highest K+ affinity. Mesenchymal cell isozymes possess an intermediate Na+ affinity and a low K+ affinity. In cancer, these ions are likely to favor optimal conditions for the function of nuclear enzymes involved in mitosis, especially a high intra-nuclear K+ concentration. A major and striking finding of this study was that in liver, metastasized CRC cells express the α3β1 isozyme. Thus, the α3β1 isozyme could potentially serve as a novel exploratory biomarker of CRC metastatic cells in liver.
Na, K-ATPase activity relies on the composition of its catalytic alpha, beta, and FXYD constituents, all of which are expressed as multiple isoforms (4alpha, 4beta, and 7 FXYD). We used reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to study Na, K-ATPase expression in uterine samples from nonlaboring elective and laboring emergency caesarean sections (CSs). Transcripts of alpha1 to 3, beta1 to 3, and FXYD1 isoforms were detected in all samples, but FXYD2 was only present in hysterectomy samples. Abundant immunoreactivity of alpha1 and moderate alpha2 was localized in myometrial smooth muscle and secretory glands of all groups. Smooth muscle and gland epithelia showed diffuse cytoplasmic alpha3 immunoreactivity. beta isoforms were detected in all groups but beta3 showed much denser immunoreactivity in myometrial samples taken from women in labor. In pregnancy, there was a switch in isoform expression, resulting in increased beta3 and decreased FXYD2 at the protein and messenger RNA (mRNA) levels. Na, K-ATPase isoform alterations may modulate uterine contractility during labor.
Citrate, an organic trivalent anion, is a major substrate for generation of energy in most cells. It is produced in mitochondria and used either in the Krebs' cycle or released into cytoplasm through a specific mitochondrial carriers. Citrate can also be taken up from blood through different plasma membrane transporters. In the cytoplasm, citrate can be used ultimately for fatty acid synthesis, which is increased in cancer cells. Here, we review the ways in which citrate can be transported and discuss the changes in transport and metabolism that occur in cancer cells. The primary focus is on the prostate gland, which is known to produce and release large amounts of citrate during its normal secretory function. The significant changes that occur in citrate-related metabolism and transport in prostate cancer are the second focus. This review strives to relate these mechanisms to molecular biology on the one hand and to clinical applications on the other.
The prostate gland is unique in its ability to secrete large amounts of zinc and citrate, suggesting that it employs unusual transport mechanisms. Intracellular ionic homeostasis in prostate is likely to be mediated by the Na,K-pump, yet there have been few studies of its regulation in this tissue. Accordingly, we explored the expression of the Na,K-pump in PC3 cells, an established cell line of human prostate epithelial cells. Total RNA from confluent monolayers of PC3 cells was isolated, reverse transcribed, and the resulting complementary DNA was amplified by polymerase chain reaction using primers specific for each of the pump's constituent subunits. The amplification revealed a complex pattern of Na,K-pump expression, with detection of mRNAs encoding the alpha1-, alpha3-, alpha4-, betal-, beta2- and beta3-isoforms. We next examined the effect on pump activity of prolactin, an important mediator of cell proliferation in prostate cancer. Monolayers exposed to 10 nM prolactin for 24 hr revealed an inhibition of 40% in ouabain-sensitive 86Rb+ uptake, a sensitive measure of pump-mediated transport. These experiments suggest that the unique transport properties of prostate may depend, at least in part, on a complicated pattern of Na,K-pump expression and regulation.
Electrophysiological characterization of normal human prostate epithelial cells showed exogenous trivalent citrate transport (release) to be K(+)-dependent.
(1) Ussing chamber recordings of short circuit current (SCC) were used to study citrate transport in the same (PNT2-C2) cell line grown on micro-pore filters as a monolayer. (2) Release of endogenous citrate from confluent cultures and tubules and segments of rat prostate was measured using a fluorescence technique. (3) Enzyme-spectrophotometry was employed to detect citrate release from segments of rat prostate.
Citrate transport across the PNT2-C2 monolayer was asymmetrical, consistent with release into the lumen-side. Fluorescence and/or enzyme-spectrophotometric measurements showed that time-dependent citrate release (endogeneous and preabsorbed) occurred from rat prostate (tubules and segments), but not kidney or lung. The release was dependent on extracellular K(+) but not Na(+).
Citrate release from prostatic cells and tissues (rat and human) was K(+)-dependent, consistent with the previous electrophysiological data.
The Na,K-ATPase comprises a family of isozymes that catalyze the active transport of cytoplasmic Na+ for extracellular K+ at the plasma membrane of cells. Isozyme diversity for the Na,K-ATPase results from the association of different molecular forms of the alpha (alpha1, alpha2, alpha3, and alpha4) and beta (beta1, beta2, and beta3) subunits that constitute the enzyme. The various isozymes are characterized by unique enzymatic properties and a highly regulated pattern of expression that depends on cell type, developmental stage, and hormonal stimulation. The molecular complexity of the Na,K-ATPase goes beyond its alpha and beta isoforms and, in certain tissues, other accessory proteins associate with the enzyme. These small membrane-bound polypeptides, known as the FXYD proteins, modulate the kinetic characteristics of the Na,K-ATPase. The experimental evidence available suggests that the molecular and functional heterogeneity of the Na,K-ATPase is a physiologically relevant event that serves the specialized functions of cells. This article focuses on the functional properties, regulation, and the biological relevance of the Na,K-ATPase isozymes as a mechanism for the tissue-specific control of Na+ and K+ homeostasis.
There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the a subunit, and at least two of the b subunit. The a subunit is the catalytic subunit. The b subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of b subunit isoforms modifies enzyme kinetics, im- plying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase b subunit isoforms b1 and b2. These antibodies, along with antibodies against the a subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of a and b isoform distribution with double- label staining demonstrated that there was no preferential as- sociation of particular a subunits with particular b subunits, nor was there an association with excitatory or inhibitory neuro- transmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether b1 and b2 are specific for neurons and glia, respec- tively, has been controversial, but expression of both b subunit types was seen here in granule cells. In rat cerebellar astro- cytes, in sections and in culture, a2 expression was prominent, yet the expression of either b subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution under- scores the subtlety of its regulation and physiological role in excitable cells.
The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged subunits in intact oocytes and protease protection assays show that the peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The peptide does not influence the formation and cell surface expression of functional Na,K-ATPase – complexes. On the other hand, the peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. subunits do not associate with individual or subunits but only interact with assembled, transport-competent – complexes. Finally, electrophysiological measurements indicate that the peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the subunit of Na,K-ATPase.
This review summarizes our experiments on the significance of the -subunit in the functional expression of Na+/K+-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na+/K+-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na+/K+-ATPase by Na+/K+- and H+/K+-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K+-ATPase -subunit and from Ile89 to the COOH–terminus of the H+/K+-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na+/K+-ATPase.
Growth factors induce the sequential expression of cellular genes whose products are thought to mediate long-term responses
to the growth factors. In mouse 3T3 fibroblastic cells, the first genes to be expressed (immediate-early genes) are activated
within minutes after the addition of platelet-derived growth factor, fibroblast growth factor, or serum. By cDNA cloning,
we have identified genes that are activated after a delay of a few hours and several hours prior to serum-induced DNA replication.
Activation of these delayed early response genes requires new protein synthesis, presumably the synthesis of immediate-early
transcription factors described previously. Partial or complete sequencing of 13 different delayed early cDNAs, representing
about 40% of the 650 primary cDNA isolates, revealed that 8 were related to known gene sequences and 5 were not. Among the
former are cDNAs encoding nonhistone chromosomal proteins [HMGI(Y) and HMGI-C], adenine phosphoribosyltransferase (APRT),
a protein related to human macrophage migration inhibitory factor (MIF), a protein of the major intrinsic protein (MIP) family
homologous to the integral membrane protein of human erythrocytes, and cyclin CYL1. In 3T3 cells, the delayed early gene response
to growth factors appears to be at least as complex as the immediate-early gene response previously described.
We examined the importance of the Na+, K(+)-ATPase in cisplatin (DDP) accumulation in 2008 human ovarian carcinoma cells and describe changes in the Na+, K(+)-ATPase in DDP-resistant cells with DDP accumulation defects. Approximately 50% of DDP accumulation was inhibitable by ouabain. DDP accumulation into 2008 cells could be maximally inhibited when cells were preincubated with ouabain for 1 h prior to DDP exposure. The half-maximal inhibition was obtained with 0.13 microM ouabain. Similar inhibition of DDP accumulation was obtained when the Na+, K(+)-ATPase was blocked by ATP depletion or by incubating cells in K(+)-free medium. This same percentage of DDP accumulation was Na+ dependent and varied directly with Na+ concentration. These effects on DDP accumulation could be detected as early as 1 min after the imposition of 0-trans conditions, strongly suggesting that the inhibition was due to modulation of a drug influx step. The Na+, K(+)-ATPase in 2008/DDP cells had a similar KD for ouabain binding and 36% less Na+, K(+)-ATPase molecules/mg of protein than 2008 cells. 2008/DDP cells 2.3 +/- 0.2 (SE, n = 3) fold cross-resistant to ouabain in a continuous exposure clonogenic assay. Despite these changes in the Na+, K(+)-ATPase, the net basal Na+, K(+)-ATPase activity was the same in sensitive and DDP-resistant cells as determined by ouabain-inhibitable 86Rb+ influx. The basal Na+ levels were also similar in the sensitive and resistant cells. These data suggest that DDP accumulation is partially Na+ dependent and that, therefore, the Na+, K(+)-ATPase which maintains the Na+ gradient may play an important role in determining how much DDP enters cells. Whether there is a causal link between the changes in the Na+, K+-ATPase in DDP-resistant cells and their DDP accumulation defect is not yet known.
Molecular genetic evidence indicates that there should be three different (Na⁺ + K⁺)-stimulated ATPase (Na,K-ATPase) α subunit isozymes in the brain where previously only two (“α” and “α(+)”) were resolved as proteins. To detect and identify α1, α2, and α3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., and Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284–288) were analyzed by gel electrophoresis and proteolytic fingerprinting. Synthetic α1 comigrated with tissue α1, while α2 and α3 comigrated with the leading and trailing edges, respectively, of tissue “α(+).” Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[³⁵S]methionine indicated the presence of α1 and α3, and a low level of α2.
Monoclonal antibodies were characterized by the electrophoretic mobility of their antigens and by their ability to recognize the Na,K-ATPases of kidney, brain, and skeletal muscle. The antibodies were used to assess isozyme expression in the brain. All three isozymes increased in abundance during development from the 18-day fetus to the adult. Small changes were seen in the relative level of expression of α1 and α3 at different developmental ages, while α2 expression increased markedly between the neonate and adult. In adult brain, all three isozymes were found in all brain regions examined. We conclude that all three isozymes are expressed as proteins and that their expression and distribution must be under complex control. No single developmental age or macroscopic brain region provides an exclusive source of any of the isozymes.
cDNA encoding the alpha-subunit of the (Na+ + K+)-ATPase was cloned from a chicken kidney cDNA library and the nucleotide sequence determined. The deduced amino acid sequence showed 92% sequence homology with the alpha-subunit of the sheep kidney (Na+ + K+)-ATPase, and high cross-species homologies were found among nucleotide sequences both in the 5'- and 3'-untranslated regions of the "kidney-type" alpha-subunit mRNAs. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk- cells. Expression of the avian alpha-sub-unit could be activated by culture of the cells in 10 mM butyrate. Cells expressing avian alpha-subunits displayed high-affinity ouabain binding (KD = 2.6 +/- 0.7 x 10(-7) M) and ouabain-sensitive 86Rb+ uptake, characteristic of avian cells.
A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.
A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.
We report evidence of the apical localization of the two Na,K-ATPase beta-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na,K-ATPase beta 1 and beta 2 subunits. These polyclonal rabbit antisera were raised against truncated beta-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the beta 1 isoform and five bands in the beta 2 isoform immunoblots. In E15 rat embryos, the beta 1 isoform was detected in brain, heart and kidney and the beta 2 isoform only in brain. While beta-subunit mRNA expression (Watts AG, Sanchez-Watts G, Emanuel JR, Levenson R (1991) Proc Natl Acad Sci USA 88, 7425-7429), immunoblotting and enzymatic activity have been determined (Zlokovic BV< mackic JB, Wang L, McComb JG, McDonough A (1993) J Biol Chem 268, 8019-8025), very little is known about the specific localization of each beta-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the beta 1 and beta 2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus.
The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.
The ion-transporting H,K-ATPase and Na,K-ATPase enzymes are each composed of an alpha and a beta subunit. It is known that assembly of the alpha and beta subunits of the Na,K-ATPase is necessary for the cell-surface delivery of the active enzyme. We have examined the molecular domains involved in the assembly of the H,K-ATPase and Na,K-ATPase alpha and beta subunits by expressing individual subunits and subunit chimeras in transiently transfected COS-1 cells. Our results demonstrate that the H,K-ATPase alpha subunit requires its beta subunit for efficient cell-surface expression, as determined by indirect immunofluorescence. The H,K-ATPase beta protein appears to be able to get to the cell surface unaccompanied by any alpha subunit and appears to localize as well to a population of intracellular vesicles. We find that a transfected chimera encoding the NH2-terminal half of the H,K-ATPase alpha subunit and the COOH-terminal half of the Na,K-ATPase alpha subunit appears to assemble with the endogenous Na,K-ATPase beta subunit and to reach the plasmalemma. Transfection of the complementary alpha chimera requires coexpression with the H,K-ATPase beta subunit in order to attain surface delivery. Thus, it is the COOH-terminal half of the alpha subunit that specifies assembly with a particular beta subunit.
Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.
The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.
The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (alpha) that traverse the membrane several times and contain the binding sites for ATP and cations. In this family, only Na,K- and H,K-ATPases have been shown to have a second subunit, a single-span glycoprotein called beta. Recently a new isoform (beta3) has been identified in mammals. Here we describe structural features and tissue distribution of the beta3 protein, utilizing an antiserum specific for its N terminus. beta3 was the only beta detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that beta3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the beta1 and beta2 subunits. Detection of beta subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of beta3/alpha ratio. The proportion of beta3 protein in the rat was highest in lung and testis. It was also present in liver and skeletal muscle, whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in beta3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes in expression. The substantial expression in lung and adult liver very likely explains long-standing puzzles about an apparent paucity of beta subunit in membranes or in discrete cellular or subcellular structures.
The Na-K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its alpha- and beta-subunits. At present, as many as four different alpha-polypeptides (alpha1, alpha2, alpha3, and alpha4) and three distinct beta-isoforms (beta1, beta2, and beta3) have been identified in mammalian cells. The stringent constraints on the structure of the Na pump isozymes during evolution and their tissue-specific and developmental pattern of expression suggests that the different Na-K-ATPases have evolved distinct properties to respond to cellular requirements. This review focuses on the functional properties, regulation, and possible physiological relevance of the Na pump isozymes. The coexistence of multiple alpha- and beta-isoforms in most cells has hindered the understanding of the roles of the individual polypeptides. The use of heterologous expression systems has helped circumvent this problem. The kinetic characteristics of different Na-K-ATPase isozymes to the activating cations (Na+ and K+), the substrate ATP, and the inhibitors Ca2+ and ouabain demonstrate that each isoform has distinct properties. In addition, intracellular messengers differentially regulate the activity of the individual Na-K-ATPase isozymes. Thus the regulation of specific Na pump isozymes gives cells the ability to precisely coordinate Na-K-ATPase activity to their physiological requirements.
The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.
Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1β (IL-1β). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto-oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to inhibit bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K+-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.
BACKGROUND
The strongly metastatic MAT-LyLu and the weakly metastatic AT-2 rat prostatic cancer cell lines have been shown to express voltage-gated ion channels differentially. In the present study, the possible contribution of voltage-gated ion channel activity to the proliferation of these cell lines was investigated, in a comparative approach.METHODS
Several voltage-gated ion channel modulators were tested for their effects on proliferation over 54 hr, using an in vitro assay. The modes of action of the chemicals were monitored by electrophysiological (patch-clamp) recording.RESULTSThe voltage-gated K+ channel blockers 4-aminopyridine (4-AP; 2 mM), margatoxin (5 nM), charybdotoxin (4.5 nM), and verapamil (50 μM) inhibited the K+ channels of both cell lines by between 38–65% and reduced the proliferation of the AT-2 cell line, in a dose-dependent manner, by 8–51%. However, only 4-AP reduced proliferation of the MAT-LyLu cell line. Tetrodotoxin (6 μM) blocked completely the voltage-gated Na+ channel expressed selectively in the MAT-LyLu cell line, but had no effect on the proliferation of either cell line. On the other hand, the presumed Na+ channel “opener” veratridine (10–50 μM) reduced significantly, in a dose-dependent manner, the proliferation of both cell lines by up to ∼30%.CONCLUSIONS
We conclude that the mechanism(s) controlling the proliferation of the weakly metastatic AT-2 cells involves voltage-gated K+ channels. In contrast, the proliferation of strongly metastatic MAT-LyLu cells is much less dependent upon voltage-gated K+ channel activity. Prostate 44:61–76, 2000. © 2000 Wiley-Liss, Inc.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K
D
values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.
1.1. A method is described for the isolation of a plasma membrane fraction from rat ventral prostate. This fraction is greatly enriched in (Na+, K+)-ATPase but also contains a small amount of Mg2+-ATPase and 5′-nucleotidase activities.2.2. The activity ratio () of the plasma membrane (Na+, K+)-ATPase was 1.7. The enzyme system specifically requires ATP as substrate and is inhibited by Ca2+ or ouabain.
Expression of Na+ channel protein was analysed in established cell lines of rat and human prostatic carcinoma origin by flow cytometry using a fluorescein-labelled polyclonal antibody. In many cell lines examined, the obtained frequency distribution profiles were bimodal and identified a subpopulation of cells which expressed high levels of Na+ channel protein. A significant positive correlation was demonstrated between the proportion of channel-expressing cells and the functional ability of individual cell lines to invade a basement membrane matrix in vitro. In addition, two transfectant cell lines containing rat prostate cancer genomic DNA were found to express significantly elevated levels of Na+ channel protein when compared with the original benign recipient cell line. Enhanced Na+ channel expression by two metastatic derivatives of these transfectant cells directly correlated with increased invasiveness in vitro. These studies strongly support the hypothesis that expression of Na+ channel protein and the metastatic behaviour of prostatic carcinoma cells are functionally related, either by endowing the membranes of these cells with specialised electrophysiological properties (e.g. enhancing their motility and/or secretory activities) and/or by perturbing endogenous mechanisms regulating ionic homeostasis within the cells.
1. A method is described for the isolation of a plasma membrane fraction from rat ventral prostate. This fraction is greatly enriched in (Na-", K-+)-ATPase but also contains a small amount of Mg-2+-ATPase and 5'-nucleotidase activities. 2. The activity ratio (Mg-2+ plus Na-+ plus K-+/Mg-2+ or Mg-+ plus Na-+) of the plasma membrane (Na-+, K-+)-ATPase was 1.7. The enzyme system specifically requires ATP as substrate and is inhibited by Ca-2+ or ouabain.
The pivotal role of the cell nucleus in androgenic control of target organs, such as the prostate, has become increasingly suspect. Equally qualified receptor activities have been found in the cytosol, endoplasmic reticulum, and plasma membrane. It is presently difficult to explain how a sex steroid can manage proliferation, metabolism, biosynthesis and secretion, all through chromatin-directed signals. In my search for a more satisfactory mediator of androgen action, I discovered that the sodium-potassium-dependent ATPase of the prostate plasma membrane binds androgen, and is activated by the hormone's presence to serve as a metabolic pacemaker. This paper is my terminal status report on one aspect of this hypothesis; namely, the nature and site of androgen binding, with clues as to the mode of action. SDS-PAGE indicates that androgen can be bound to the beta-subunit of prostatic Na,K-ATPase. Selective enrichment of the enzyme by reversible coupling to either concanavalin A or a DHT-affinity column support this conclusion. Several studies show the dynamic effect of androgen binding: increased ouabain binding; enhancement of this binding by facilitated phosphorylation; spectroscopic evidence of conformational shifts, possibly consequences of these suggested activities for regulation, especially of metabolism, are examined.
Accumulation and secretion of extraordinarily high levels of citrate are principal functions of the prostate gland of humans and other animals. To achieve this, prostate secretory cells must possess unique metabolic relationships which distinguish them from virtually all other cells. Furthermore, citrate metabolism is markedly altered in benign prostatic hyperplasia (BPH) and in prostatic carcinoma (CA). This review assimilates existing information and presents current concepts related to 1) the pathway of metabolism associated with net citrate production, 2) the involvement of transporting mechanisms associated with citrate secretion, 3) energy implications of citrate production, 4) altered metabolic relationships in BPH and CA, and 5) the importance of citrate relationships as biochemical markers for characterizing prostate secretory epithelial cells. It is hoped that this review will bring attention to the importance and urgency of elucidating and understanding the metabolic relationships associated with citrate production by normal and neoplastic prostate epithelial cells. Research in these areas has been severely neglected despite the fact that the combined incidence of BPH and CA constitutes the most prevalent neoplastic disease among men.
The transport of aspartate was measured in isolated prostate cells. Two kinetically distinct uptake systems of high (Km = 10.0 microM) and low (Km = 0.8 mM) affinity were observed. Transport by both systems was Na+ dependent and saturable. The tissue distribution of aspartate was also determined by measuring the washout kinetics of endogenous and radiolabeled aspartate from prostate fragments. The distribution of aspartate resulted in 47% of aspartate in the intracellular compartment and 52% in the extracellular (presumably) lumenal compartment. The distribution of aspartate resulted in a calculated intracellular to plasma concentration gradient of approximately 43. We concluded that prostate cells maintain a considerable intracellular to plasma gradient for aspartate and that these cells contain a transport mechanism capable of uptake of aspartate against this considerable gradient.
The ionic composition of human prostatic fluid varied greatly between individuals, reflecting the secretory activity of the gland and the presence or absence of prostatic inflammatory disease. In normal prostatic fluid the major anion was citrate, while chloride concentrations were lower. Their counterions were mainly sodium and potassium, together with calcium, magnesium and zinc. Prostatic secretions from men with prostatitis comprised mainly sodium and chloride. The electrolytes were closely correlated to each other (except for sodium, which was essentially invariant at about 145 nm). The molar changes per mole of citrate were about 0.52, potassium; -0.53, chloride; 0.17, calcium; 0.14, magnesium; and 0.09, zinc. The pH was also associated with citrate, decreasing from 8.0 to 6.2 as the citrate increased. These various ionic changes can be explained as responses to citrate secretion, without the need to propose specific transport mechanisms for the other ions measured. The marked effect of prostatic inflammation on the composition of prostatic fluid can be seen as being due mainly to decreased secretion rather than active modification.
The purposes of this study were: (1) to determine if a (Na⁺ + K⁺)-dependent, ouabain-sensitive, androgen-responsive ATPase is present in the microsomal fraction of the human prostate as it is in the rat ventral prostate; (2) to determine the degree and nature of interaction and interdependence of the ATPase with the steroid-binding and steroid-metabolizing activities of the prostate and, also, with the histological characteristics of the gland. The results indicate that ATPase which shows particular responsiveness to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and dehydroepiandrosterone is present. The microsomes, which contain this enzymic activity, show relatively high affinity for the active steroids and for oestradiol-17β. The intrinsic steroid-sensitive, cation-dependent ATPase activity varies widely from gland to gland in parallel with the steroid binding and 3α-hydroxysteroid dehydrogenase activity. In comparisons of the enzymatic complements of glands with different histological features, the concentration of 4-en-3-oxosteroid-5α-reductase activity in tissue showing predominantly epithelial hyperplasia was greater than that in normal or carcinomatous glands or glands with stromal hypertrophy. The possible role of the ATPase as a mediator of hormonal stimulation, and the implications of the findings to an understanding of the development of benign prostatic hypertrophy, are discussed.
The membrane ATPase of prostatic microsomes was further examined in order to better characterize its interaction with testosterone. Steroid treatment increased the rate of the cation-dependent ATPase at all concentrations of MgATP from 0.25–10 μM, maximum velocities of both treated and untreated enzyme occurring at 5 μM. The androgenized microsomes were more temperature sensitive also. A 10-degree rise in temperature (25–35 C) produced a significantly greater increase in the ATPase activity of the treated than of the control preparation. Tests of the two components of the ATPase process revealed that the androgen was ineffective upon the Na⁺-dependent phosphorylation of the enzyme but produced a significant acceleration of the K⁺-dependent dephosphorylation. Means to explain how the steroid regulates the catalytic and vectorial actions of the enzyme are discussed.
Glutamate dehydrogenase activity was determined in mitochondrial preparations from rat ventral prostate and rat kidney. Kinetic parameters of the ventral prostate enzyme were comparable to those for the kidney enzyme. Glutamate dehydrogenase activity in the direction of glutamate oxidative deamination was inhibited by alpha-ketoglutarate. However, the characteristics of alpha-ketoglutarate inhibition indicated that glutamate oxidation via glutamate dehydrogenase can occur at in vivo prostatic alpha-ketoglutarate levels. These results suggest that glutamate dehydrogenase activity in prostate may provide a continuous source of alpha-ketoglutarate for aspartate transamination to oxalacetate and ultimate citrate synthesis. In addition prostate mitochondria are able to couple the glutamic dehydrogenase reaction to aspartate aminotransferase. Under these conditions aspartate in the presence of glutamate and acetyl coenzyme A will result in a net synthesis of citrate. Consequently we propose an aspartate-glutamate pathway for citrate synthesis in prostate.
Previously described vimentin monoclonal antibodies recognize other cellular or intermediate filament proteins in addition to vimentin. In contrast the set of murine monoclonal antibodies described here and elicited using porcine vimentin as antigen appear specific for vimentin. All seven antibodies react only with vimentin in "immunoblot" analysis on gel electrophoretically separated polypeptides from total cell extracts. They do not recognize other closely related intermediate filament proteins including desmin and GFA (glial fibrillary acidic protein). Immunofluorescence microscopy on tissue sections shows a positive reaction in connective tissue, endothelial cells, vascular smooth muscle cells and astrocytes. Typical intermediate filament profiles are documented in fibroblasts and in other cultured cells known to contain vimentin. Although all antibodies react with human and porcine vimentin some show species-specific restriction when rat, mouse and chicken vimentin are used. These results, together with different IgG types and certain aspects of the staining patterns, allow a subdivision of the antibodies. The vimentin antibodies described here complete a set of monoclonal antibodies each specific for only one intermediate filament protein type. Their reaction on human material allows their use in pathology to determine the histogenetic origin of neoplasms.
The effects of organic-solvent extracts of Urtica dioica (Urticaceae) on the Na+,K(+)-ATPase of the tissue of benign prostatic hyperplasia (BPH) were investigated. The membrane Na+,K(+)-ATPase fraction was prepared from a patient with BPH by a differential centrifugation of the tissue homogenate. The enzyme activity was inhibited by 10(-4)-10(-5) M of ouabain. The hexane extract, the ether extract, the ethyl acetate extract, and the butanol extract of the roots caused 27.6-81.5% inhibition of the enzyme activity at 0.1 mg/ml. In addition, a column extraction of stinging nettle roots using benzene as an eluent afforded efficient enzyme inhibiting activity. Steroidal components in stinging nettle roots, such as stigmast-4-en-3-one, stigmasterol, and campesterol inhibited the enzyme activity by 23.0-67.0% at concentrations ranging from 10(-3)-10(-6) M. These results suggest that some hydrophobic constituents such as steroids in the stinging nettle roots inhibited the membrane Na+,K(+)-ATPase activity of the prostate, which may subsequently suppress prostate-cell metabolism and growth.
Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.
Clinically-used drugs such as furosemide, bumetanide and cardiac glycosides, are modulators of transmembrane fluxes of cations. Recently, it has been suggested that the regulation of intracellular cation concentrations could be a primary target for anti-neoplastic drugs, and that the cytotoxic activity may be altered by inhibitors of cation fluxes at the level of the plasma membrane. Therefore, we investigated the mechanisms by which cations are translocated across the plasma membrane of malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines. The interactions between cation flux inhibitors and the cytotoxicity of estramustine were also evaluated. Ouabain, the classical inhibitor of Na+, K+ATPase, markedly reduced 86Rb (K+) influx in all three lines, indicating that this ion transport system is present in the cells. Furosemide and especially bumetanide inhibited the 86Rb influx, indicating the presence of the Na+, K+, Cl- co-transport system. The potassium channel blocker, tetraethylammonium, but not apamin reduced the influx of 86Rb showing that high conductance K+ channels are present, but that channels of low conductance probably do not exist in these cell lines. The Na+, K+, Cl- co-transport inhibitors furosemide and bumetanide significantly reduced cytotoxicity of estramustine in P31 cells, whereas no interaction between other K+ flux inhibitors and the anti-neoplastic drugs were detected in any of the cell lines investigated. Thus, the data show that Na+, K+, ATPase and NA+, K+, Cl- co-transport systems and K+ channels of high conductance are present in malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines, and that inhibition of the Na+, K+, Cl- co-transport system in P31 is associated with reduced cytotoxicity of estramustine. The results justify further studies evaluating the role of these cation flux pathways in terms of targets for anti-neoplastic therapy.
Prostate secretory epithelial cells have the unique and highly specialized function of accumulating and secreting extraordinarily high levels of citrate. Aspartate is a major four-carbon source of oxaloacetate required for the net synthesis of citrate by these cells. The prostate epithelial cells contain a Na(+)-coupled, high-affinity aspartate transporter which permits the cells to accumulate aspartate from circulation against a large concentration gradient. Because of this unique relationship between aspartate and citrate, it was important to determine if citrate modulated the high-affinity transport of aspartate into prostate epithelial cells. The current studies with freshly prepared prostate epithelial cells obtained from rat ventral prostate demonstrated that citrate exerted two effects on aspartate transport. Physiological levels of extracellular citrate (i.e., equivalent to circulating levels) markedly inhibited (cis-inhibitory effect) aspartate transport. In contrast, intracellular citrate at concentrations associated with normal in situ cells resulted in two levels of stimulation of aspartate transport. A 4-fold increase in aspartate transport occurred when the intracellular citrate was increased up to 500 nmols/g, and an 11-fold increase resulted when the intracellular citrate concentration was elevated to 1800 nmols/g (which is uniquely characteristic of prostate cells). The combined effect of the cis-inhibitory and trans-stimulatory actions of citrate was a consistent 1-2 fold increase in aspartate transport. These studies support the concept that citrate is a physiological regulator of the high-affinity transport of aspartate into prostate secretory epithelial cells in association with the unique and highly specialized function of net citrate production and secretion.
The prostate gland produces and secretes extraordinarily high levels of citrate. Studies with rat ventral prostate (VP) have demonstrated that aspartate can serve as a four-carbon source of oxalacetate in the synthesis of citrate. To achieve this, prostate secretory epithelial cells must contain a transport system for the active uptake of aspartate from circulation. The present studies with VP epithelial cells confirm the existence of a Na+ -dependent high-affinity L-aspartate transporter. The transporter has an optimal pH ≈ 7.5 and is temperature dependent. It appears to be an anionic amino acid transporter capable of transporting L-glutamate but not basic or neutral amino acids. The transporter is inhibited by ATPase inhibitors, thereby indicating its dependency on a Na+ gradient. The characteristics of the high-affinity L-aspartate transporter are consistent with its operation at the basilar membrane for the transport of circulating aspartate into the cell.
Castration (24 hr) resulted in a significant decrease- in the ability of VP epithelial cells to transport L-aspartate. The administration of testosterone to castrated rats completely restored L-aspartate transport. In addition, in vitro testosterone addition (10−8 M for 30 min) to isolated prostate epithelial cells markedly increased L-aspartate transport. Both cycloheximide and actinomycin inhibited the testosterone effect. The studies reveal that testosterone is a regulator of this Na+ -dependent high-affinity L-aspartate transporter. The mechanism of this testosterone effect appears to involve both RNA and protein synthesis. We now have a model system to elucidate this novel effect of testosterone.
Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1 beta (IL-1 beta). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto- oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K(+)-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.
Monoclonal antibodies to isoforms of the Na,K-ATPase have become important tools in the study of the enzyme's distribution, physiological roles, and gene regulation, and when their epitopes are defined, they are useful in the study of enzyme structure as well. Evidence is presented that the alpha3-specific antibody McBX3 recognizes an unusual epitope that is not present on alpha3 in the heart. The epitope, which is also found in kidney alpha1 from some species, was mapped to a site on the large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding regions from alpha3 from brain (where McBX3 recognizes alpha3) and heart demonstrated that the tissue difference in epitope is not due to alternative splicing of the mRNA. Instead, hydroxylamine sensitivity indicated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the well-characterized antibodies McK1 (alpha1) and McB2 (alpha2). The antibody XVIF9-G10 reacted with the alpha3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cellular structures in sections of the rat retina. A new alpha1-specific antibody, 6F, was characterized and mapped to another site near the N terminus; this antibody has broader species specificity than the other well-characterized alpha1 antibody, McK1.
Na+,K(+)-ATPase plays a major role in the reabsorption of sodium by the kidney. This organ is highly heterogeneous, and its functional unit, the nephron, is formed of successive epithelia with specific morphological and functional characteristics. Na+,K(+)-ATPase expression varies along the nephron. Variations in the catalytic activity of the pump, the number of active pumps expressed in the basolateral membrane, its substrate dependency towards Na and K, and its sensitivity to the inhibitor ouabain have been observed in distinct tubular segments. Most authors agree on the large (or unique) prevalence of the alpha 1 and beta 1 isoforms of the two subunits of Na+,K(+)-ATPase in each nephron segment, although at different levels. The cortical collecting duct represents a unique epithelium to study the physiological relevance of the regulation of Na+,K(+)-ATPase activity, including an immediate substrate activation, a rapid recruitment of active pumps from a reserve pool, and long-term hormonal effects. Whether these functions require other molecular determinants than the alpha 1 and beta 1 isoform subunits remains to be established.
There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the alpha subunit, and at least two of the beta subunit. The alpha subunit is the catalytic subunit. The beta subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of beta subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase beta subunit isoforms beta1 and beta2. These antibodies, along with antibodies against the alpha subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of alpha and beta isoform distribution with double-label staining demonstrated that there was no preferential association of particular alpha subunits with particular beta subunits, nor was there an association with excitatory or inhibitory neurotransmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether beta1 and beta2 are specific for neurons and glia, respectively, has been controversial, but expression of both beta subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, alpha2 expression was prominent, yet the expression of either beta subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution underscores the subtlety of its regulation and physiological role in excitable cells.
This review summarizes our experiments on the significance of the beta-subunit in the functional expression of Na+/K(+)-ATPase. The beta-subunit acts like a receptor for the alpha-subunit in the biogenesis of Na+/K(+)-ATPase and facilitates the correct folding of the alpha-subunit in the membrane. The alpha-subunit synthesized in the absence of the beta-subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the beta-subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the beta-subunit confers a resistance to trypsin on the alpha-subunit. The conformations induced in the alpha-subunit of Na+/K(+)-ATPase by Na+/K(+)- and H+/K(+)-ATPase beta-subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the beta-subunit is involved in the folding of the alpha-subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K(+)-ATPase beta-subunit and from Ile89 to the COOH-terminus of the H+/K(+)-ATPase beta-subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type, respectively. The first disulfide loop of the extracellular domain of the beta-subunits is critical in the expression of functional Na+/K(+)-ATPase.
While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEnery et al., 1991, Proc. Natl. Acad. Sci. 88, 11095-11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.
Cisplatin is the most active anticancer agent for lung cancer. It has been reported that intracellular accumulation of cisplatin is important in determining resistance to cisplatin, which may be modulated by Na+, K(+)-ATPase activity. On the other hand, it is well-known that sorbitol, a metabolite of glucose mediated by aldose reductase, reduces Na+, K(+)-ATPase in diabetic neuropathy. In this study, the effect of exogenous sorbitol on Na+, K(+)-ATPase activity and sensitivity to cisplatin was evaluated using human non-small-cell lung cancer (NSCLC) cell lines. In the NSCLC cell lines, EBC-1, PC-3, and RERF-LC-MS the cytotoxicities of cisplatin were impaired by exposure to sorbitol in these cell lines. Na+, K(+)-ATPase was inactivated and intracellular accumulation of cisplatin was decreased by the exposure. These results suggest that accumulation of sorbitol may induce resistance to cisplatin in NSCLC cells, and diabetes poorly controlled may be one of the determinants of the antitumor effect of cisplatin in NSCLC.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue-specific isoforms exist for each of these, alpha1, alpha2 and alpha3 and beta1, beta2 and beta3. We have proposed that an additional alpha isoform, alpha4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative alpha4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in alpha4 isoform cDNA transfected 3T3 cells. Using an alpha4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.
The Na+, K+-ATPase is an ubiquitous plasma membrane protein complex that belongs to the P-type family of ion motive ATPases. Under normal conditions, it couples the hydrolysis of one molecule of ATP to the exchange of three Na+ for two K+ ions, thus maintaining the normal gradient of these cations in animal cells. Despite decades of investigation of its structure and function, the structural basis for its cation specificity and for conformational coupling of the scalar energy of ATP hydrolysis to the vectorial movement of Na+ and K+ have remained a major unresolved issue. This paper summarizes our recent studies concerned with these issues. The findings indicate that regions(s) of the amino terminus and first cytoplasmic (M2/M3) loop act synergistically to affect the steady-state conformational equilibrium of the enzyme. Although carboxyl- or hydroxyl-bearing amino acids comprise the cation-binding and occlusion sites, our experiments also suggest that these interactions may be modulated by juxtapositioned cytoplasmic regions.