Microbial MIP channels (original) (raw)
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Oligomerization of water and solute channels of the major intrinsic protein (MIP) family
Kidney International, 2001
(250 to 290 amino acids), and the high conservation intrinsic protein (MIP) family. Water and small solute fluxes throughout the MIP family may indicate a common fold: through cell membranes are ensured in many tissues by seleca NH 2 cytosolic portion followed by a hydrophobic stretch tive pores that belong to the major intrinsic protein family of six transmembrane helices ). Among highly (MIP). This family includes the water channels or aquaporins conserved amino acids in the family, two repetitions of (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of Asp, Pro, Ala residues (NPA box) localized in the B and representatives of each subfamily. Following solubilization in E loops draw the sequence signature of the family (Fig. the nondenaturing detergents n-octyl-glucoside (OG) and Tri-1B). The folding of these two loops in the membrane ton X-100 (T-X100), AQPs remain in their native homotetrabilayer is proposed to be responsible of the pore formameric state, while GlpF always behaves as a monomer. Solute facilitators are fully solubilized by the detergent N-lauroyl sartion and solute movement across the membrane [2, 3]. cosine (NLS), while AQPs are not. Analyses of mutants and Interestingly, AQPs are widely distributed in bacteria, chimeras demonstrate a close correlation between the water plants, and animals, while GlpFs have been characterized transport function and the resistance to NLS solubilization. only within microorganisms such as bacteria or yeast. In Thus, AQPs and solute facilitators exhibit different behaviors mammals, ten MIPs have been cloned and functionally in mild detergents; this could reflect differences in quaternary organization within the membranes. We propose that the oligocharacterized. Some of them, such as AQP1, AQP3, and merization state or the strength of self-association is part of the AQP4, are widely distributed in the body [4]. In contrast, mechanisms used by MIP proteins to ensure solute selectivity.
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 specu- late 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.
Members of the superfamily of major intrinsic proteins (MIPs) facilitate water and solute permeability across cell membranes and are found in sources ranging from bacteria to humans. Aquaporin and aquaglyceroporin channels are the prominent members of the MIP superfamily. Experimental studies show that MIPs are involved in important phys- iological processes in mammals and plants. They are implicated in several human diseases and are considered to be attractive drug targets for a wide range of diseases such as cancer, brain edema, epilepsy, glaucoma, and congestive heart failure. Three-dimensional structures of MIP channels from diverse sources reveal that MIPs adopt a unique conserved hourglass helical fold consisting of six transmembrane heli- ces (TM1–TM6) and two half-helices (LB and LE). Conserved NPA motifs near the center and the aromatic/arginine selectivity filter (Ar/R SF) toward the extracellular side consti- tute two narrow constriction regions within the channel. Structural knowledge com- bined with simulation studies have helped to investigate the role of these two constriction regions in the transport and selectivity of the solutes. With the availability of many genome sequences from diverse species, a large number of MIP genes have been identified. Homology models of 1500 MIP channels have been used to derive structure-based sequence alignment of TM1–TM6 helices and the two half-helices LB and LE. Thirteen residues are highly conserved in different transmembrane helices and half-helices. High group conservation of small and weakly polar residues is observed in 27 positions at the interface of two interacting helices. Thus, although the MIP sequences are diverse, the hourglass helical fold is maintained during evolution with the conservation of these 40 positions within the transmembrane region. We have pro- posed a generic structure-based numbering scheme for the MIP channels that will facil- itate easier comparison of the MIP sequences. Analysis of Ar/R SF in all 1500 MIPs indicates the extent of diversity in the four residues that form this narrow region. Certain residues are completely avoided in the SF, even if they have the same chemical nature as that of the most frequently observed residues. For example, arginine is the most pre- ferred residue in a specific position of Ar/R SF, whereas lysine is almost always avoided in any of the four positions. MIP channels with highly hydrophobic or hydrophilic Ar/R SF have been identified. Similarly, there are examples of MIP channels in which all four res- idues of Ar/R SF are bulky, thus almost occluding the pore. Many plant MIPs possess small residues at all SF positions, resulting in a larger pore diameter. A majority of MIP channels are yet to be functionally characterized, and their in vivo substrates are not yet identified. A complete understanding of the relationship between the nature of Ar/R SF and the solutes that are transported is required to exploit MIP channels as potential drug targets.
Structural basis of water-specific transport through the AQP1 water channel
Nature, 2001
Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction 1,2. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as the aquaporins (AQP): AQP0-AQP9, which can be divided into two major groups 3,4 ; AQP0, AQP1, AQP2, AQP4-AQP6 and AQP8, permeable to water but not to small organic and inorganic molecules 5 , and AQP3, AQP7 and AQP9, permeable to glycerol or urea as well as water 3,5. AQP1 (M.W. 28 kDa) was initially found in red blood cells and renal proximal tubules 2,6. AQP1 water channels allow water, but not ions including protons, to freely and bidirectionally move across the cell membrane 5,7. Sequence analysis shows high homology among members of the AQP1 family and that the two halves of the sequence exhibit a high degree of similarity 4. Each sequence half contains an NPA (asn, pro, ala) motif which is conserved throughout the AQP superfamily including the glycerol facilitators. Moderate resolution projection and low resolution 3-D maps of AQP1 derived from electron crystallographic studies provided the first structure based evidence for a general architecture consisting of six helices surrounding two putative helical structures within the membrane bilayer 8-11. Models of AQP1 derived from electron crystallographic structural studies at about 4 Å resolution have recently been reported and independently confirmed the presence of two non-membrane spanning helices 12-14 .
European Journal of Biochemistry, 1990
The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with CAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible CAMP-dependent protein kinase phosphorylation sites. Lens membranes, whch contain 50 -60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21 -22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239 -259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with CAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.
A new subfamily LIP of the major intrinsic proteins
BMC Genomics, 2014
Background: Proteins of the major intrinsic protein (MIP) family, or aquaporins, have been detected in almost all organisms. These proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules. Results: We compared the predicted amino acid sequences of 20 MIPs from several algae species of the phylum Heterokontophyta (Kingdom Chromista) with the sequences of MIPs from other organisms. Multiple sequence alignments revealed motifs that were homologous to functionally important NPA motifs and the so-called ar/R-selective filter of glyceroporins and aquaporins. The MIP sequences of the studied chromists fell into several clusters that belonged to different groups of MIPs from a wide variety of organisms from different Kingdoms. Two of these proteins belong to Plasma membrane intrinsic proteins (PIPs), four of them belong to GlpF-like intrinsic proteins (GIPs), and one of them belongs to a specific MIPE subfamily from green algae. Three proteins belong to the unclassified MIPs, two of which are of bacterial origin. Eight of the studied MIPs contain an NPM-motif in place of the second conserved NPA-motif typical of the majority of MIPs. The MIPs of heterokonts within all detected clusters can differ from other MIPs in the same cluster regarding the structure of the ar/R-selective filter and other generally conserved motifs. Conclusions: We proposed placing nine MIPs from heterokonts into a new group, which we have named the LIPs (large intrinsic proteins). The possible substrate specificities of the studied MIPs are discussed.
Structure of Functional Single AQP0 Channels in Phospholipid Membranes
Journal of Molecular Biology, 2003
Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Twodimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (,73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.