The molecular basis of renal tubular transport disorders

Sodium and water homeostasis are key to the survival of organisms. Reabsorption of sodium and water occurs throughout the tubule structure of the nephron, the basic functional unit of the kidney, by various transport mechanisms. Altered transport protein function can lead to renal tubular disorders resulting in metabolic alkalosis, hypokalemia, hypertension, and decreased capacity to concentrate urine, for instance. However, recent advances in molecular physiology, molecular genetics and expression cloning systems have aided in unraveling the molecular basis of some renal tubular disorders. This review will examine the molecular basis of Bartter's syndrome, Gitelman's syndrome, Liddle's syndrome, and autosomal nephrogenic diabetes insipidus. An understanding of the molecular basis of these disorders of the human kidney can give us a better understanding of basic renal function of lower mammals and other vertebrates.     

PIN-pointing the molecular basis of auxin transport.

Significant advances in the genetic dissection of the auxin transport pathway have recently been made. Particularly relevant is the molecular analysis of mutants impaired in auxin transport and the subsequent cloning of genes encoding candidate proteins for the elusive auxin efflux carrier. These studies are thought to pave the way to the detailed understanding of the molecular basis of several important facets of auxin action.     

The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae

The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in α‐glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette‐transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous α‐glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX‐ and SpuA‐dependent manner. SpuA is able to degrade glycogen into a ladder of α‐1,4‐glucooligosaccharides while the high‐affinity interaction (K_a ～ 10⁶ M^?1) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X‐ray crystallographic analyses of apo‐ and complexed MalX illuminate the protein's specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal α‐glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaeα‐glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.     

Estradiol-stimulated nuclear ribonucleoprotein transport in the rat uterus: a molecular basis

The present investigation probes the intranuclear molecular changes that serve to link the nuclear binding of estradiol with the hormone-stimulated ribonucleoprotein (RNP) transport in the rat uterus. Within 2 min of in vitro exposure of isolated uterine nuclei to 10 nM 17 beta-estradiol a Mg2+-dependent nuclear ATPase becomes activated and reaches its peak activity. This is immediately followed by a phase of ATP resynthesis. This newly synthesized ATP serves as the substrate for the nuclear protein kinases. Cyclic AMP inhibits this ATP resynthesis and, as a consequence, prevents the estradiol-stimulated nuclear protein kinase activity and the exit of the RNP-estradiol complex from the nuclei. cGMP is stimulatory to the estradiol-mediated nuclear ribonucleoprotein transport.     

Phosphate transport: molecular basis, regulation and pathophysiology

Inorganic phosphate (Pi) is fundamental to cellular metabolism and skeletal mineralization. Ingested Pi is absorbed by the small intestine, deposited in bone, and filtered by the kidney where it is reabsorbed and excreted in amounts determined by the specific needs of the organism. Two distinct renal Na-dependent Pi transporters, type IIa (NPT2a, SLC34A1) and type IIc (NPT2c, SLC34A3), are expressed in brush border membrane of proximal tubular cells where the bulk of filtered Pi is reabsorbed. Both are regulated by dietary Pi intake and parathyroid hormone. Regulation is achieved by changes in transporter protein abundance in the brush border membrane and requires the interaction of the transporter with scaffolding and signaling proteins. The demonstration of hypophosphatemia secondary to decreased renal Pi reabsorption in mice homozygous for the disrupted type IIa gene underscores its crucial role in the maintenance of Pi homeostasis. Moreover, the recent identification of mutations in the type IIc gene in patients with hereditary hypophosphatemic rickets with hypercalciuria attests to the importance of this transporter in Pi conservation and subsequent skeletal mineralization. Two novel Pi regulating genes, PHEX and FGF23, play a role in the pathophysiology of inherited and acquired hypophosphatemic skeletal disorders and studies are underway to define their mechanism of action on renal Pi handling in health and disease.     

The molecular basis for Na-dependent phosphate transport in human erythrocytes and K562 cells

The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.     

A molecular basis for tungstate selectivity in prokaryotic ABC transport systems

The essential trace compounds tungstate and molybdate are taken up by cells via ABC transporters. Despite their similar ionic radii and chemical properties, the WtpA protein selectively binds tungstate in the presence of molybdate. Using site-directed mutagenesis of conserved binding pocket residues, we established a molecular basis for tungstate selectivity.     

The molecular basis of photoperiodism

The rotation of our planet results in regular changes in environmental cues such as daylength and temperature, and organisms have evolved a molecular oscillator that allows them to anticipate these changes and adapt their development accordingly. In many plants, the transition from vegetative to reproductive growth is controlled by photoperiod, which synchronises flowering with favourable seasons of the year. Here, we describe the notable progress that has been made in identifying the molecular mechanisms that measure daylength and control of flowering time in Arabidopsis, a long day (LD) plant, and in rice, a short day (SD) plant. Although the components of the Arabidopsis regulatory network seem to be conserved in other species, the difference in the function of particular genes may contribute to the reverse response to daylength observed between LD and SD plants. We also highlight the recent advances in understanding the regulatory mechanisms that underlie other developmental transitions controlled by photoperiod, including tuberisation and the onset of dormancy in the buds of perennial plants.     

The molecular basis of allorecognition in ascidians

The process of allorecognition consists of an ability to discriminate self from non-self. This discrimination is used either to identify non-self cells and reject them ("non-self histocompatibility") or to identify self cells and reject them (as in the avoidance of self-fertilization by hermaphrodites ("self incompatibility"). The molecular basis governing these two distinct systems has been studied recently in hermaphroditic ascidian urochordates. Harada et al. postulated two highly polymorphic self-incompatibility loci, Themis (A and B), that are transcribed from both strands, forward to yield sperm (s-) trans-membrane antigen, and reverse to yield the egg vitelline coat (v-) receptor. De Tomaso et al. characterized a candidate histocompatibility locus, encoding a highly variable immunoglobulin. Nyholm et al. isolated its candidate allorecognition receptor, fester. Only a minute similarity was found in the structure of the genes involved. It appears that ascidian harbor two very separate types of labeling and recognition genetic systems: one for self and the other for non-self.     

The molecular basis of fluidity in membranes

Fluidity as a prominent feature of the phospholipid portion of biological membranes, as well as of model phospholipid bilayer systems, has been detected by numerous physical techniques¹. However, correlation of this fluidity with biological functions of membranes is, as yet, documented in only a few cases. For example, fatty acid auxotrophs of E. coli grown on different fatty acids exhibit an abruptly increased rate of transport of metabolites across the cell wall at temperatures above the “melting” temperature of the fatty acid supplement^2,3). The physical properties of lipids extracted from E. coli also reflect the temperature at which the bacteria were grown⁴). Fluidity of hydrocarbon chains has been related to the calcium dependent ATPase activity of sarcoplasmic vesicles⁵). A number of other essential functions of biological membranes may very well be associated with fluidity^6,7), but such considerations are limited by lack of precise knowledge of the molecular basis of fluidity and of the rates of motions involved. The following discussion will review the use of spin labels^8–11 to determine the rates of several of the motions involved in the fluidity of phospholipid bilayers and, where possible, to provide a structural basis for these motions.     

The molecular basis of alpha-thalassaemia in Thailand

The molecular basis of alpha-thalassaemia has been established in 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome. This study has shown that in this population there are at least 18 different types of chromosome carrying seven independent alpha-thalassaemia mutations one of which is a novel deletion removing the entire alpha-globin gene complex. Although there are a limited number of alpha-thalassaemia determinants in the Thai population, there is a remarkable degree of variation in the genetic markers which flank them. These markers may be of value in establishing the evolutionary history of the alpha-thalassaemias.     

Going the distance with auxin: unravelling the molecular basis of auxin transport.

Auxin represents one of the most important classes of signalling molecules described in plants. Auxins regulate several fundamental cellular processes including division, elongation and differentiation. Indole-3-acetic acid (IAA), the principal form of auxin in higher plants, is first synthesized within young apical tissues, then conveyed to its basal target tissues by a specialized delivery system termed polar auxin transport. The polarity of IAA movement represents one of the most novel aspect of auxin signalling. IAA transport has been demonstrated to involve auxin influx and efflux carrier activities. The adoption of a mutational approach in the model plant Arabidopsis thaliana has led to the identification of a number of genes which encode components for, or regulate the activity of, the auxin transport machinery. This paper will review the advances being made in identifying and characterizing these auxin transport-related gene products and discuss their importance within the context of Arabidopsis development.