Proton‐translocating, vacuolar‐type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na+ gradients, imposed by Na+/K+ ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H+ V‐ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V‐ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V‐ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na+ uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V‐ATPases. Awareness of plasma membrane energization by V‐ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture. BioEssays 21:637–648, 1999. © 1999 John Wiley & Sons, Inc.

Slc26a9 is a recently identified anion transporter that is abundantly

MDCK cells form monolayers which have many of the properties usually found in transporting epithelia. The present article is devoted to the study of the structural and functional polarization of MDCK cells, which is one of the central features of transporting epithelia. The results show: (i) that MDCK monolayers transport 2.6 mumol hr-1 cm-2 of sodium in the apical to basolateral direction; (ii) the passive flux of this ion is relatively large (20.3 mole hr-1 cm-2), which is a characteristic of leaky epithelia; (iii) a large fraction of the penetration of sodium into the cells proceeds through an amiloride-sensitive channel, and the exit is operated mainly by a ouabain-sensitive pump; (iv) the net transport of sodium from the apical to the basolateral side agrees with the asymmetric labeling of the pumps with 3H-ouabain; (v) this asymmetric labeling agrees, in turn, with a higher concentration of intramembrane particles (IMPs) in freeze-fracture replicas of the basolateral side of the plasma membrane; (vi) the structural polarization of confluent MDCK cells is also revealed by the location of microvilli, occluding junctions, and pinocytotic vesicles; and (vii) the presence of a continuous ring formed by actin microfilaments visualized by immunofluorescence under the lateral aspect of the plasma membrane that may be related to the distribution of the occluding junctions, which act as barriers separating apical from basolateral membrane components.

SLC26 family members are anionic transporters involved in Cl^- and HCO₃^- absorption or secretion in epithelia. SLC26A9, preferentially expressed in the lung, is a poorly characterized member of this family. In this study, we investigated the transport properties of human SLC26A9 to determine its functional and pharmacological characteristics. SLC26A9 protein expression results in the appearance of an anionic current exhibiting an apparently linear current/voltage relationship and increases in ³⁶Cl influxes and effluxes. The sequences of conductivity, Cl^- >I^- > NO₃^- ≧ gluconate > SO₄ ^2- and selectivity (P_x/P_CI), I^- > NO₃^- > Cl^- > gluconate > SO₄^2- are found. Cl^- channel inhibitors DIDS and NS 3623 inhibit SLC26A9 associated currents while the specific CFTR inhibitor (CFTR(inh)-172) or glybenclamide has little effect. Elevation of intracellular cAMP (a CFTR activator) is also ineffective whereas increasing intracellular calcium blocks the SLC26A9 associated currents. The HCO₃^- conductance mediated by the SLC26A9 protein expression is low and no intracellular pHi changes are detectable under conditions favoring a Cl^-/HCO₃^- exchange. However, the presence of HCO₃^-/CO₂ stimulates the Cl^--transporting activity of SLC26A9 in Xenopus laevis oocytes or SLC26A9-transduced COS-7 cells. As an important initial step in characterizing SLC26A9 function, we conclude that SLC26A9 is a Cl^- channel and we suggest that HCO₃^- acts as a modulator of the channel. SLC26A9 physiological role in airway epithelia and its potential interaction with CFTR remain to be elucidated.

We investigated the possible functional- and physical protein-interactions between two airway Cl(-) channels, SLC26A9 and CFTR. Bronchial CFBE41o- cell lines expressing CFTR(WT) or CFTR(ΔF508) were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole-cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR(WT) and CFTR(ΔF508) cell lines. In CFBE41o- cells and CFBE41o-/CFTR(WT) cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)-stimulated currents were enhanced in SLC26A9-transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR((inh)-172) or GlyH-101 inhibition in both groups). In CFBE41o-/CFTR(ΔF508) cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK-stimulated currents and level of CFTR((inh)-172) inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK-stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK-stimulated currents was not found in oocytes co-expressing SLC26A9 and CFTR(ΔF508). In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR(ΔF508). Our co-immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9-STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents.