Protean PTEN: form and function

Germline mutations distributed across the PTEN tumor-suppressor gene have been found to result in a wide spectrum of phenotypic features. Originally shown to be a major susceptibility gene for both Cowden syndrome (CS), which is characterized by multiple hamartomas and an increased risk of breast, thyroid, and endometrial cancers, and Bannayan-Riley-Ruvalcaba syndrome, which is characterized by lipomatosis, macrocephaly, and speckled penis, the PTEN hamartoma tumor syndrome spectrum has broadened to include Proteus syndrome and Proteus-like syndromes. Exon 5, which encodes the core motif, is a hotspot for mutations likely due to the biology of the protein. PTEN is a major lipid 3-phosphatase, which signals down the PI3 kinase/AKT pro-apoptotic pathway. Furthermore, PTEN is a protein phosphatase, with the ability to dephosphorylate both serine and threonine residues. The protein-phosphatase activity has also been shown to regulate various cell-survival pathways, such as the mitogen-activated kinase (MAPK) pathway. Although it is well established that PTEN's lipid-phosphatase activity, via the PI3K/AKT pathway, mediates growth suppression, there is accumulating evidence that the protein-phosphatase/MAPK pathway is equally important in the mediation of growth arrest and other crucial cellular functions.     

Anion exchanger 1 in red blood cells and kidney: Band 3's in a pod

The bicarbonate/chloride exchanger 1 (AE1, Band 3) is abundantly expressed in the red blood cell membrane, where it is involved in gas exchange and functions as a major site of cytoskeletal attachment to the erythrocyte membrane. A truncated kidney isoform (kAE1) is highly expressed in type A intercalated cells of the distal tubules, where it is vital for urinary acidification. Recently, kAE1 has emerged as a novel physiologically significant protein in the kidney glomerulus. This minireview will discuss the known interactions of kAE1 in the podocytes and the possible mechanisms whereby this important multispanning membrane protein may contribute to the function of the glomerular filtration barrier and prevent proteinuria.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

Structure and function of the human Na+/H+ exchanger isoform 1

Sodium proton exchangers (NHEs) constitute a large family of polytopic membrane protein transporters found in organisms across all domains of life. They are responsible for the exchange of protons for sodium ions. In archaea, bacteria, yeast and plants they provide increased salt tolerance by removing sodium in exchanger for extracellular protons. In humans they have a host of physiological functions, the most prominent of which is removal of intracellular protons in exchange for extracellular sodium. Human NHE is also involved in heart disease, cell growth and in cell differentiation. NHE’s physiological roles and the intriguing pathological consequences of their actions, make them a very important target of structural and functional studies. There are nine isoforms identified to date in humans. This review provides a brief overview of the human NHE’s physiological and pathological roles and cellular/tissue distribution, with special attention to the exemplar member NHE1. A summary of our knowledge to date of the structure and function of NHE1 is included focusing on a discussion of the recent discrepancies reported on the topology of NHE1. Finally we discuss a newly discovered relative of the NHE1 isoform, the Na+/Li+ exchanger, focusing on its predicted topology and its potential roles in disease.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.     

阴离子交换蛋白家族的结构与功能

阴离子交换蛋白（anion exchanger,AE）家族由AE1、AE2和AE3三个成员组成,介导哺乳动物细胞普遍存在的C1一／HCO,跨膜交换过程,进而调节细胞内pH值（intracellular pH,pHi）和细胞体积。在极化的上皮细胞,Cl^-／HCO3^-的跨膜交换也是经上皮的酸碱分泌和重吸收的重要调节因子。AE1丰富地表达在红细胞,其N末端截短形式也表达在肾脏;AE2广泛地表达在各种组织,但以胃最为丰富;AE3表达在脑、视网膜和心脏。近年来对AE家族结构与功能的研究取得的新进展揭示了AE家族在某些病理过程中发挥重要作用。本文就AE家族的结构与功能及其病理作用进行了讨论。     

Mucins: structure, function, and associations with malignancy.

Mucins are a family of high molecular weight, highly glycosylated glycoproteins found in the apical cell membrane of human epithelial cells from the mammary gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus and rete testis. Increased synthesis of the core protein and alterations in the carbohydrates attached to these glycoproteins are believed to play important roles in the function and proliferation of tumour cells. Aberrant glycosylation leads not only to the production of novel carbohydrate structures, but also to the exposure of the core peptide. These novel epitopes may be candidates for diagnosis or therapy, by using either synthetic mucin fragments as vaccines, or monoclonal antibody-based reagents which detect these structures.     

Anion exchanger 1b, but not sodium-bicarbonate cotransporter 1b, plays a role in transport functions of zebrafish H+-ATPase-rich cells

Similar to mammalian proximal tubular cells, H(+)-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na(+) uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na(+)/HCO(3)(-) cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na(+) or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na(+) content and the skin H(+) activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na(+) uptake/acid secretion mechanisms in zebrafish HR cells.     

Protean Literacy: Extending the Discourse on Empowerment.

Protean Literacy: Extending the Discourse on Empowerment. Concha Delgado-Gaitan. Washington, D.C.: Falmer Press, 1996. 151 pp.     

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

Peak exercise stroke volume: associations with cardiac structure and diastolic function.

One of the most debilitating effects of primary aging is the decline in aerobic exercise capacity. One of its causes is an age-related decline in peak exercise stroke volume. This study's main purpose was to determine the cardiovascular adaptations to aging that most influence peak exercise stroke volume in the elderly. We hypothesized that increased left ventricular (LV) filling and mild concentric LV remodeling would be associated with an increase in peak exercise stroke volume corrected for lean body mass (LBM) and that an increased augmentation index (AI), which is a marker of arterial stiffness, would be associated with a decrease. A second aim was to determine the adaptations to aging that most influence LV concentric remodeling in the elderly. We hypothesized that AI would be a predictor of LV mass/LBM and the LV posterior wall thickness-to-LV radius ratio (h/r). We performed a cross-sectional study of cardiac and vascular adaptations to aging in 52 sedentary, elderly subjects. LV filling [as measured by the early-to-late transmitral flow velocity ratio (E/A)] was inversely correlated with and was an independent predictor of peak exercise stroke volume/LBM and was also a predictor of LV remodeling. AI was a predictor of LV remodeling (LV mass/LBM) but not of peak exercise stroke volume/LBM. We conclude that 1) maintenance of LV filling (E/A <1) is associated with a higher peak exercise stroke volume/LBM in very elderly subjects and thus may be a useful adaptation that enhances stroke volume during peak exercise, 2) LV remodeling and AI are less influential on peak exercise stroke volume/LBM, and 3) AI was the most important predictor of LV remodeling.     

Ion selectivity of colicin E1: III. Anion permeability

The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 Å. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N,N-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 m NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions.The authors wish to thank Dr. F.S. Cohen for making available unpublished data and for helpful comments. This work was supported by National Institutes of Health grant GM 37396 and by the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Initiative (E.R.K.)     

Amoebae as Exemplary Cells: The Protean Nature of an Elementary Organism

In the nineteenth century protozoology and early cell biology intersected through the nexus of Darwin’s theory of evolution. As single-celled organisms, amoebae offered an attractive focus of study for researchers seeking evolutionary relationships between the cells of humans and other animals, and their primitive appearance made them a favourite model for the ancient ancestor of all living things. Their resemblance to human and other metazoan cells made them popular objects of study among morphologists, physiologists, and even those investigating animal behaviour. The amoeba became the exemplar of the new protoplasmic cell concept of mid-century and because its apparent simplicity made it widely generalizable it became a popular subject in a breadth of experimental investigations and theoretical speculations. It was able to do this because “the amoeba” denotes not a particular organism, but a general type of behaviour common to the cells of a range of protozoa, simple plants and higher animals. Its status as an exemplary cell also rested upon auxiliary philosophical assumptions about what constitutes a primitive characteristic and the thesis that evolution is a progressive development of order from chaos.     

Correlating structure, dynamics, and function in transmembrane segment VII of the Na/H exchanger isoform 1

We place ¹⁵N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na⁺/H⁺ exchanger isoform 1. Although G261-S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six ¹⁵N labelled residues examined in this study all have similar dynamics on the ps-ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the μs-ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261-A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.     

AIDS dementia and HIV-1-induced neurotoxicity: Possible pathogenic associations and mechanisms

AIDS Dementia Complex (ADC) is a syndrome of cognitive, behavioral, and motor deficits resulting from HIV-1 infection within the brain. ADC is characterized by variable degrees of neuronal cell death and gliosis that likely result, at least, in part from release of metabolic products, cytokines, and viral proteins from infected macrophages, although a unifying explanation for the neurological dysfunction has yet to be established. Major unanswered questions include: (i) do neurologic symptoms result from neuronal cell death and/or dysfunction in surviving neurons?; (ii) are viral genomic sequences determinants of neurotoxicity?; (iii) is HIV infection of neurons and astrocytes relevant to pathogenesis?, and (iv) what circulating factors within the brain affect neuronal cell survival and function? This review addresses the association between HIV-1 replication within the brain, production of potential neurotoxins and possible mechanisms of induction of neurotoxicity and neuronal dysfunction contributing to the pathogenesis of ADC.     

The Na+/H+ exchanger cytoplasmic tail: structure,function, and interactions with tescalcin

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.