Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine

An isoform of the mammalian renal type II Na/Pi-cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na/Pi-cotransporter. Na/Pi-cotransport mediated by the type IIb cotransporter was studied in oocytes of Xenopus laevis. The results indicate that type IIb Na/Pi-cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na/Pi-cotransporter represents a candidate for small intestinal apical Na/Pi-cotransport.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302→Ala and Arg-302→Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H+ movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H+ translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.

Long-range transport of mineral dust in the global atmosphere: Impact of African dust on the environment of the southeastern United States

Soil dust is a major constituent of airborne particles in the global atmosphere. Dust plumes frequently cover huge areas of the earth; they are one of the most prominent and commonly visible features in satellite imagery. Dust is believed to play a role in many biogeochemical processes, but the importance of dust in these processes is not well understood because of the dearth of information about the global distribution of dust and its physical, chemical, and mineralogical properties. This paper describes some features of the large-scale distribution of dust and identifies some of the geological characteristics of important source areas. The transport of dust from North Africa is presented as an example of possible long-range dust effects, and the impact of African dust on environmental processes in the western North Atlantic and the southeastern United States is assessed. Dust transported over long distances usually has a mass median diameter <10 μm. Small wind-borne soil particles show signs of extensive weathering; consequently, the physical and chemical properties of the particles will greatly depend on the weathering history in the source region and on the subsequent modifications that occur during transit in the atmosphere (typically a period of a week or more). To fully understand the role of dust in the environment and in human health, mineralogists will have to work closely with scientists in other disciplines to characterize the properties of mineral particles as an ensemble and as individual particles especially with regard to surface characteristics.

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria1

The induction of a high-affinity state of the CO2-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO2 concentrations were resuspended in low-CO2 buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO2-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO2 and HCO3− fluxes indicated that only the affinity of HCO3− transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO2 uptake remained unchanged. Fast induction of high-affinity HCO3− transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO3− transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO3− transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.