Transmembrane helix 5 is critical for the high water permeability of aquaporin

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major role in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topology of six transmembrane helices connected by five loops (loops A-E). Despite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2. When expressed in Xenopus oocytes, the osmotic water permeability (P(f)) of MIP and AQP2 was 48 and 245 x 10(-)(4) cm/s, respectively. Substitutions in loops B-D failed to increase P(f), whereas substitution of loop E significantly increased P(f) 1.5-fold. A similar increase in P(f) was observed with the substitution of the front half of loop E. P(f) measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However, P(f) values of the loop E chimeras were only approximately 30% of that of AQP2. Simultaneous exchanges of loop E and a distal half of transmembrane helix 5 just proximal to loop E increased P(f) to the level of that of AQP2. Replacement of helix 5 alone stimulated P(f) 2.7-fold. Conversely, P(f) was decreased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P(f) was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spliced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We speculate that this portion contributes to the formation of the aqueous pore and the determination of the flux rate.     

A water channel of the nematode C.elegans and its implications for channel selectivity of MIP proteins

A genome project focusingon the nematode Caenorhabditis elegans has demonstrated thepresence of eight cDNAs belonging to the major intrinsic proteinsuperfamily. We functionally characterized one of these cDNAs namedC01G6.1. Injection of C01G6.1 cRNA increased the osmotic waterpermeability (P_f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed toincrease the glycerol permeability. It has been speculated that the MIPfamily may be separated into two large subfamilies based on thepresence or absence of two segments of extra amino acid residues (~15amino acids) at the second and third extracellular loops. BecauseC01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with thoseof AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that ofwild-type AQP-CE1, although the values of P_f andurea permeability were decreased by 39-74% and 28-65%,respectively. These results suggest that the two segments of extraamino acid residues may not contribute to channel selectivity orformation of the route for small solutes.

     

Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site.

The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha-TIP showed that the TIPs are differentially expressed.     

Mercurial sensitivity of aquaporin 1 endofacial loop B residues

The water channel protein aquaporin-1 (AQP1) has two asparagine-proline-alanine (NPA) repeats on loops B and E. From recent structural information, these loops are on opposite sides of the membrane and meet to form a pore. We replaced the mercury-sensitive residue cysteine 189 in AQP1 by serine to obtain a mercury-insensitive template (C189S). Subsequently, we substituted three consecutive cysteines for residues 71-73 near the first NPA repeat (76-78) in intracellular loop B, and investigated whether they were accessible to extracellular mercurials. AQP1 and its mutants were expressed in Xenopus laevis oocytes, and the osmotic permeability (P(f)) of the oocytes was determined. C189S had wild-type P(f) but was not sensitive to HgCl(2). Expression of all three C189S cysteine mutants resulted in increased P(f), and all three mutants regained mercurial sensitivity. These results, especially the inhibitions by the large mercurial p-chloromercunbenzene-sulfonic acid (pCMBS) ( approximately 6A wide), suggest that residues 71-73 at the pore are accessible to extracellular mercurials. A 30-ps molecular dynamics simulation (at 300 K) starting with crystallographic coordinates of AQP1 showed that the width of the pore bottleneck (between Connolly surfaces) can vary (w(avg) = 3.9 A, sigma = 0.75; hydrated AQP1). Thus, although the pore width would be > or = 6 A only for 0.0026 of the time, this might suffice for pCMBS to reach residues 71-73. Alternative explanations such as passage of pCMBS across the AQP1 tetramer center or other unspecified transmembrane pathways cannot be excluded.     

Molecular and functional characterization of a vasotocin-sensitive aquaporin water channel in quail kidney

Both mammals and birds can concentrate urine hyperosmotic to plasma via a countercurrent multiplier mechanism, although evolutionary lines leading to mammals and birds diverged at an early stage of tetrapod evolution. We reported earlier (Nishimura H, Koseki C, and Patel TB. Am J Physiol Regul Integr Comp Physiol 271: R1535-R1543, 1996) that arginine vasotocin (AVT; avian antidiuretic hormone) increases diffusional water permeability in the isolated, perfused medullary collecting duct (CD) of the quail kidney. In the present study, we have identified an aquaporin (AQP) 2 homolog water channel in the medullary cones of Japanese quail, Coturnix coturnix (qAQP2), by RT-PCR-based cloning techniques. A full-length cDNA contains an 822-bp open reading frame that encodes a 274-amino acid sequence with 75.5% identity to rat AQP2. The qAQP2 has six transmembrane domains, two asparagine-proline-alanine (NPA) sequences, and putative N-glycosylation (asparagine-124) and phosphorylation sites (serine-257) for cAMP-dependent protein kinase. qAQP2 is expressed in the membrane of Xenopus laevis oocytes and significantly increased its osmotic water permeability (P(f)), inhibitable (P < 0.01) by mercury chloride. qAQP2 mRNA (RT-PCR) was detected in the kidney; medullary mRNA levels were higher than cortical levels. qAQP2 protein that binds to rabbit anti-rat AQP2 antibody is present in the apical/subapical regions of both cortical and medullary CDs from normally hydrated quail, and the intensity of staining increased only in the medullary CDs after water deprivation or AVT treatment. The relative density of the approximately 29-kDa protein band detected by immunoblot from the medullary cones was modestly higher in water-deprived/AVT-treated quail. The results suggest that 1) medullary CDs of quail kidneys express a mercury-sensitive functioning qAQP2 water channel, and 2) qAQP2 is at least partly regulated by an AVT-dependent mechanism. This is the first clear identification of AQP2 homolog in nonmammalian vertebrates.     

Two distinct aquaporin-4 cDNAs isolated from medullary cone of quail kidney

Water deprivation or arginine vasotocin upregulates aquaporin-2 (AQP2) expression in apical and subapical regions of medullary collecting duct (CD) cells of Coturnix coturnix quail (q) kidneys. We therefore aimed to determine whether the CD has AQPs mediating water exit from the intracellular to the extracellular (interstitial) space. Using a homologue cloning technique, we isolated two distinct qAQP4 cDNAs from quail medullary cones; long (L, open reading frames) and short (S) cDNA encoded 335 (qAQP4-L) and 301 (qAQP4-S) amino acids with, respectively, 80% and 87% identity to human long- and short-form AQP4. qAQP4-S is identical to qAQP4-L from the second initiation site. Both isoforms have two NPA motifs, but lack cysteine at the known mercury-sensitive site. qAQP4-L and qAQP4-S are expressed in membranes of Xenopus laevis oocytes, but both failed to increase the water permeability (P(f)) of oocytes exposed to a hypotonic solution. Glutamate (Q242) replacement with histidine did not increase P(f). With conventional RT-PCR and real-time PCR, qAQP4-L/S mRNA signals were detected in the brain, lung, heart, intestine, adrenal gland, skeletal muscle, liver, and kidney (higher in medulla than in cortical region). qAQP4-L mRNA was detected only in the brain and adrenal gland. Orthogonal arrays of intramembranous particles were not detected in quail CDs. The results suggest that although qAQP4-L and qAQP4-S have high homology to mammalian AQP4, their physiological function may be different.     

Transport of water and glycerol in aquaporin 3 is gated by H(+).

Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L(p), was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. The L(p) values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeability P(Gl), obtained from [(14)C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (sigma). For AQP3, water and glycerol interacted by competing for titratable site(s): sigma(Gl) was 0.15 at neutral pH but doubled at pH 6.4. The sigma values were smaller for polyols in which the -OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol(-1). We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites.     

Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes

We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.     

Developmental regulation of the direct interaction between the intracellular loop of connexin 45.6 and the C terminus of major intrinsic protein (aquaporin-0)

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.     

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.     

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10–15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.     

Water transport in aquaporins: osmotic permeability matrix analysis of molecular dynamics simulations

Single-channel osmotic water permeability (p(f)) is a key quantity for investigating the transport capability of the water channel protein, aquaporin. However, the direct connection between the single scalar quantity p(f) and the channel structure remains unclear. In this study, based on molecular dynamics simulations, we propose a p(f)-matrix method, in which p(f) is decomposed into contributions from each local region of the channel. Diagonal elements of the p(f) matrix are equivalent to the local permeability at each region of the channel, and off-diagonal elements represent correlated motions of water molecules in different regions. Averaging both diagonal and off-diagonal elements of the p(f) matrix recovers p(f) for the entire channel; this implies that correlated motions between distantly-separated water molecules, as well as adjacent water molecules, influence the osmotic permeability. The p(f) matrices from molecular dynamics simulations of five aquaporins (AQP0, AQP1, AQP4, AqpZ, and GlpF) indicated that the reduction in the water correlation across the Asn-Pro-Ala region, and the small local permeability around the ar/R region, characterize the transport efficiency of water. These structural determinants in water permeation were confirmed in molecular dynamics simulations of three mutants of AqpZ, which mimic AQP1.     

Cloning of a new aquaporin (AQP10) abundantly expressed in duodenum and jejunum

A new aquaporin (AQP10) was identified in human small intestine. This gene encoded a 264-amino-acid protein with high sequence identity with AQP3 (53%), 9 (52%), and 7 (43%). These AQPs constitute one subfamily of AQP family that is differentiated from the other subfamily of AQP (AQP0, 1, 2, 4, 5, 6, and 8) by sequence homology. Ribonuclease protection assay and Northern blotting demonstrated almost exclusive expression of AQP10 mRNA in the duodenum and jejunum. In situ hybridization localized it in absorptive jejunal epithelial cells. Xenopus oocytes expressing AQP10 exhibited an increased osmotic water permeability in a mercury-sensitive manner. Although AQP10 belongs to the AQP subfamily, which has been characterized by permeability to water and neutral solutes such as urea and glycerol, it was not permeable to urea nor glycerol. The specific expression of AQP10 suggests its contribution to the water transport in the upper portion of small intestine.     

Aquaporin-4 gene disruption in mice reduces brain swelling and mortality in pneumococcal meningitis

The astroglial water channel aquaporin-4 (AQP4) facilitates water movement into and out of brain parenchyma. To investigate the role of AQP4 in meningitis-induced brain edema, Streptococcus pneumoniae was injected into cerebrospinal fluid (CSF) in wild type and AQP4 null mice. AQP4-deficient mice had remarkably lower intracranial pressure (9 +/- 1 versus 25 +/- 5 cm H2O) and brain water accumulation (2 +/- 1 versus 9 +/- 1 microl) at 30 h, and improved survival (80 versus 0% survival) at 60 h, through comparable CSF bacterial and white cell counts. Meningitis produced marked astrocyte foot process swelling in wild type but not AQP4 null mice, and slowed diffusion of an inert macromolecule in brain extracellular space. AQP4 protein was strongly up-regulated in meningitis, resulting in a approximately 5-fold higher water permeability (P(f)) across the blood-brain barrier compared with non-infected wild type mice. Mathematical modeling using measured P(f) and CSF dynamics accurately simulated the elevated lower intracranial pressure and brain water produced by meningitis and predicted a beneficial effect of prevention of AQP4 upregulation. Our findings provide a novel molecular mechanism for the pathogenesis of brain edema in acute bacterial meningitis, and suggest that inhibition of AQP4 function or up-regulation may dramatically improve clinical outcome.     

Molecular cloning and expression of aquaporin 1 [correction of aquapolin 1] (AQP1) in dog kidney and erythroblasts

Complementary DNA of the water channel aquaporin 1 (AQP1) was cloned from dog kidney and erythroblasts. The cDNA amplified from mRNA in dog kidney was 816 bp, the same as that in bovines, but longer by 6 bp than that in humans, mice and rats. The 235-bp fragment cDNA amplified from the mRNA in dog erythroblasts, which was differentiated from peripheral blood, was completely identical to the corresponding sequence of cDNA from the dog kidney. Thus, mature red blood cells from dog may have AQP1 in their cell membranes. The amino acid sequence in dog AQP1 was 91-94% identical to that in the other species mentioned above. Dog AQP1 has six predicted transmembrane domains, two NPA motifs, one mercury-sensitive site and four consensus phosphorylation sites, the same as the other species. However, dog and bovine AQP1 have only one N-glycosylation site, while two glycosylation sites were found in human and rodent AQP1. Xenopus oocytes injected with the mRNA of the dog AQP1 exhibited high water permeability in a hyposmotic medium. Thus, dog AQP1 performs water transport the same as in the other species.     

Aquaglyceroporins,one channel for two molecules

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.

BACKGROUND INFORMATION: Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys(189) as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys(189) of AQP1. Indeed, the osmotic water permeability (P(f)) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. RESULTS: The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys(178), which is located at loop D facing the intracellular side, is a target responding to Hg2+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. CONCLUSIONS: Our results suggest that mercury inhibits the P(f) of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.     

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.