Si permeability of a deficient Lsi1 aquaporin in tobacco can be enhanced through a conserved residue substitution.

Silicon (Si) is a beneficial substrate for many plants, conferring heightened resilience to environmental stress. A plant's ability to absorb Si is primarily dependent on the presence of a Si-permeable Lsi1 (NIP2-1) aquaporin in its roots. Structure-function analyses of Lsi1 channels from higher plants have thus far revealed two key molecular determinants of Si permeability: (a) the amino acid motif GSGR in the aromatic/arginine selectivity filter and (b) 108 amino acids between two highly conserved NPA domains. Curiously, tobacco (Nicotiana sylvestris) stands as a rare exception as it possesses an Lsi1 (NsLsi1) with these molecular signatures but is reported as a low Si accumulator. The aim of this study was therefore to identify whether additional determinants influence Si permeability via Lsi1 channels, focusing on the role of residues that differ uniquely in NsLsi1 relative to functional Lsi1 homologs. We observed tobacco indeed absorbed Si poorly (0.1% dw), despite NsLsi1 being expressed constitutively in planta. Si influx measured in NsLsi1-expressing Xenopus oocytes was very low (<13% that of OsLsi1 from rice (Oryza sativa) over a 3-hr time course), which likely explains why tobacco is a low Si accumulator. Interestingly, NsLsi1P125F displayed a significant gain of function (threefold increase in Si influx relative to NsLsi1WT), which coincided with a threefold increase in plasma membrane localization in planta, as measured by transient expression of GFP constructs in Nicotiana benthamiana leaves. These findings thus reveal a novel molecular determinant of Si transport in plants and inform breeding, biotechnological, and agricultural practices to effectively utilize Si in the context of plant resilience to environmental stress.

Endocytic recycling of Na+ -K+ -Cl- cotransporter type 2: importance of exon 4.

KEY POINTS: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 27-exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load. It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL. In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization. Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation-Cl- cotransporter family member. ABSTRACT: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 12-transmembrane (TM) domain cell surface glycoprotein that is expressed in the thick ascending limb (TAL) of Henle and stimulated during cell shrinkage. It comes as three splice variants (A, B and F) that are identical to each other except for TM2 and the following connecting segment (CS2). Yet, these variants do not share the same localization, transport characteristics and physiological roles along the TAL. We have recently found that while cell shrinkage could exert its activating effect by increasing NKCC2 expression at the cell surface, the variants also responded differentially to this stimulus. In the current work, a mutagenic approach was exploited to determine whether CS2 could play a role in carrier trafficking and identify the residues potentially involved. We found that when the residue of position 238 in NKCC2A (F) and NKCC2B (Y) was replaced by the corresponding residue in NKCC2F (V), carrier activity increased by over 3-fold and endocytosis decreased concomitantly. We also found that when the residue of position 230 in NKCC2F (M) was replaced by the one in NKCC2B (T), carrier activity and affinity for ions both increased substantially whereas expression at the membrane decreased. Taken together, these results suggest that CS2 is involved in carrier trafficking and that two of its residues, those of positions 238 and 230, are part of an internalization motif. They also indicate that the divergent residue of position 230 plays the dual role of specifying ion affinity and sustaining carrier internalization.

Regulation of Na+-K+-Cl- cotransporter type 2 by the with no lysine kinase-dependent signaling pathway.

Na+-K+-Cl- cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl-] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.

A new gold standard approach to characterize the transport of Si across cell membranes in animals.

Silicon (Si) is increasingly recognized as an essential trace element in animals, especially since the identification of mammalian Si transport systems and Si responsive genes not long ago. During many years, however, efforts to gain substantial insight into the biological role of this element in animals have achieved partial success due in part to the unavailability of validated protocols to study Si movement across biological membranes. To circumvent such limitations, we have developed a general transport assay in which cellular Si content was determined by automated electrothermal atomic absorption spectrophotometry. We have found this assay to provide great analytic sensitivity with Si detection thresholds of less than 1 µM, that is, below or very close to the concentration range of animal cells. We have also found this assay to provide valid and cost-effective determinations in Si transport studies while requiring workable quantities of samples. The protocol described here should thus become gold standard toward accelerated progress in the field of Si transport.

Phosphoregulation of K+ -Cl- cotransporters during cell swelling: Novel insights.

The K+ -Cl- cotransporters (KCCs) belong to the cation-Cl- cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl- ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl- ] (Cli ), but the mechanisms at play are still ill-defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine-deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1-related proline/alanine-rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C-terminus of full-length KCC4 led to higher levels of heterologous K+ -Cl- cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric-sensitive kinase-dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.

Identification of key residues involved in Si transport by the aquaglyceroporins.

We recently demonstrated that the aquaglyceroporins (AQGPs) could act as potent transporters for orthosilicic acid (H4SiO4). Although interesting, this finding raised the question of whether water and H4SiO4, the transportable form of Si, permeate AQGPs by interacting with the same region of the pore, especially in view of the difference in molecular radius between the two substrates. Here, our goal was to identify residues that endow the AQGPs with the ability to facilitate Si diffusion by examining the transport characteristics of mutants in which residues were interchanged between a water-permeable but Si-impermeable channel (aquaporin 1 [AQP1]) and a Si-permeable but water-impermeable channel (AQP10). Our results indicate that the composition of the arginine filter (XX/R), known to include three residues that play an important role in water transport, may also be involved in Si selectivity. Interchanging the identities of the nonarginine residues within this filter causes Si transport to increase by approximately sevenfold in AQP1 and to decrease by approximately threefold in AQP10, whereas water transport and channel expression remain unaffected. Our results further indicate that two additional residues in the AQP arginine filter may be involved in substrate selectivity: replacing one of the residues has a profound effect on water permeability, and replacing the other has a profound effect on Si permeability. This study has thus led to the identification of residues that could play a key role in Si transport by the AQGPs and shown that substrate selectivity is likely ensured by more than one checkpoint within or near the pore.

Ablation of Potassium-Chloride Cotransporter Type 3 (Kcc3) in Mouse Causes Multiple Cardiovascular Defects and Isosmotic Polyuria.

Inactivation of Kcc3 in a mixed 129/Sv×C57BL/6 mouse background has been previously found to increase systemic blood pressure (BP) through presumed neurogenic mechanisms. Yet, while this background is generally not considered ideal to investigate the cardiovascular system, KCC3 is also expressed in the arterial wall and proximal nephron. In the current study, the effects of Kcc3 ablation was investigated in a pure rather than mixed C57BL/6J background under regular- and high-salt diets to determine whether they could be mediated through vasculogenic and nephrogenic mechanisms. Aortas were also assessed for reactivity to pharmacological agents while isolated from the influence of sympathetic ganglia. This approach led to the identification of unforeseen abnormalities such as lower pulse pressure, heart rate, aortic reactivity and aortic wall thickness, but higher diastolic BP, left ventricular mass and urinary output in the absence of increased catecholamine levels. Salt loading also led systolic BP to be higher, but to no further changes in hemodynamic parameters. Importantly, aortic vascular smooth muscle cells and cardiomyocytes were both found to express KCC3 abundantly in heterozygous mice. Hence, Kcc3 inactivation in our model caused systemic vascular resistance and ventricular mass to increase while preventing extracellular fluid volume to accumulate. Given that it also affected the physiological properties of aortas in vitro, vasculogenic mechanisms could therefore account for a number of the hemodynamic abnormalities observed.

Aquaporins Mediate Silicon Transport in Humans.

In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport.

Cation-Cl- cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl(-)-coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid-polyamine-organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA-amino acid transport at the surface of HEK-293 cells. We have also found that the influx of PAs in CCC9a-expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl-. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation.

Phosphoregulation of K(+)-Cl(-) cotransporter 4 during changes in intracellular Cl(-) and cell volume.

It has long been stated that the K(+)-Cl(-) cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N-ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C(VOL)) and intracellular [Cl(-)] ([Cl(-)](i)) on the activity and phosphorylation levels (P(LEV)) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)-sensitive effectors. We found that (1) low [Cl(-)](i) or low C(VOL) leads to decreased activity but increased P(LEV), (2) high C(VOL) leads to increased activity but no decrease in P(LEV) and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P(LEV). Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl(-)](i) or C(VOL) modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A-sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787-796, 2009. (c) 2009 Wiley-Liss, Inc.

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters.

Little is known regarding the quaternary structure of cation-Cl- cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl- cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl- cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl- cotransport achieves higher levels of functional diversity than foreseen.