Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-prolinealanine (NPA) motifs, the central constriction, and an aromatic͞ arginine (ar͞R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar͞R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A͞ R195V), did not affect water permeability. The double mutant AQP1-H180A͞R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A͞H180A) enlarged the maximal diameter of the ar͞R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar͞R constriction in AQP1-R195V and AQP1-H180A͞R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar͞R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar͞R constriction. mutational analysis ͉ proton filter ͉ solute selectivity O rthodox aquaporins (AQPs) constitute one branch of water-conducting channels within the superfamily of major intrinsic proteins (1). In recent years, the protein structure of the prototypical mammalian AQP1 has been refined to 2.2-Å resolution (2-5). Two highly conserved structural features within the channel were proposed as filters that exclude the passage of solutes larger than water and of charged molecules, including protons. A central constriction is formed by the capping amino acids Asn-Pro-Ala (NPA constriction) at each positive end of two short ␣-helices, such that the two NPA motifs are pinched in the center of the pore. Proline and alanine are exchangeable to some extent, whereas asparagine is invariable (1). Extensive molecular dynamics͞quantum mechanical simulations suggest that the free energy barrier located at the NPA constriction predominates in the exclusion of protons (reviewed in ref. 6). Depending on the computational approach, a second significant energy barrier, termed aromatic͞arginine (ar͞R) constriction, exists. It is located below the channel mouth and is even narrower than the central NPA constriction (6, 7). It is formed by four amino acids (Phe-56, His-180, Cys-189, and Arg-195 in rat AQP1). The ensemble of Phe, His, and Arg is highly conserved in orthodox AQPs. Their side chains directly flank the pore, whereas the less-conserved C...

In this study the plane elasticity problem for a nonhomogeneous layer containing a crack perpendicular to the boundaries is considered. It is assumed that the Young’s modulus of the medium varies continuously in the thickness direction. The problem is solved under three different loading conditions, namely fixed grip, membrane loading, and bending applied to the layer away from the crack region. Mode I stress intensity factors are presented for embedded as well as edge cracks for various values of dimensionless parameters representing the size and the location of the crack and the material nonhomogeneity. Some sample results are also given for the crack-opening displacement and the stress distribution.

The aquaporin protein family generally seems to be designed for the selective passage of water or glycerol. Charged molecules, metal ions and even protons are strictly excluded. Recently, particular aquaporin isoforms were reported to conduct unconventional permeants, i.e., the unpolar gases carbon dioxide and nitric oxide, the polar gas ammonia, the oxidative oxygen species hydrogen peroxide, and the metalloids antimonite, arsenite and silicic acid. Here, we summarize the available data on permeability properties and physiological settings of these aquaporins and we analyze which structural features might be connected to permeability for non-water, non-glycerol solutes.

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a ϳ35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH 4 ؉ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake. Ammonium transporters (AMTs)1 of the AMT/MEP/Rh protein family have been identified in all domains of life, including plants, bacteria, archea, yeast, and animals (1, 2). AMT/ MEP/Rh proteins are highly hydrophobic membrane proteins with a predicted molecular mass of ϳ45-55 kDa and 11 or 12 putative transmembrane spans. Initially AMT/MEP/Rh ammonium transporters from yeast and plants were identified molecularly by functional complementation of a yeast mutant defective in ammonium uptake (3-5). Later, homologs were isolated from bacteria (6) and animals (Caenorhabditis elegans), and phylogenetic analysis showed that mammalian Rh (rhesus) blood group polypeptides belong to the same superfamily (7). Heterologously expressed RhAG and a homolog from kidney (RhGK ϭ RhCG) were also shown to function as ammonium transporters (8, 9).Plants require transporters for NH 4 ϩ acquisition from a wide range of external concentrations and are able to concentrate and transiently accumulate NH 4 ϩ in the cytosol before being metabolized or further compartmentalized (10). Ammonium transport across root plasma membranes is biphasic, consisting of a high-affinity and a low-affinity nonsaturating component (11,12). The high-affinity transport system, which operates predominantly at low external ammonium concentrations, is energized by the membrane potential. In tomato, the NH 4 ϩ -uniporter LeAMT1;1 encodes a component of the high-affinity transport system that depends on the membrane potential (13). The molecular identity of the low-affinity transport system, however, is less clear. It contributes significantly to overall NH 4 ϩ uptake at higher external ammonium concentrations (Ͼ1 mM) and may have a distinct transport mechanism, because uncharged NH 3 or charged NH 4 ϩ may be the substrate (11, 12, 14,...