Created by Sarah Elkin
The high water permeability characteristic of mammalian red cell membranes is known to be caused by the protein AQP1(1). 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(6). AQP1 is distributed in apical and basolateral membranes of renal proximal tubules and descending thin limbs as well as capillary endothelia. 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 aquaporins (AQP): AQP0-AQP9(6). The aquaporin of interest is bovine aquaporin 1 (AQP1: PDB ID=1J4N), a water channel protein. AQP1 has an isoelectic point of 6.58 and a molecular weight of 28800.48 g/mol. AQP1 contains an associated B-nonylglucoside ligand. Several of the aquaporins have consensus sequences for N-linked glycosylation and are monoglycosylated in native tissues. Biochemical analysis of purified human erythrocyte AQP1 indicated that 50% of AQP1 monomers are glycosylated with a polylactosaminyl oligosaccharide of 5.4 kDa in the first extracellular loop (10). However, glycosylation does not appear to be important for aquaporin function or membrane targeting (7).
The water channel in which this protein is found consists of three topological elements, an extracellular and a cytoplasmic vestibule, connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine-182, which is conserved among all known water-specific channels, is critical in establishing water specificity(6).
Aquaporin-1 from the Canis lupus familiaris was found to have a similar sequence to bovine Aquaporin-1. The BLAST results, such as the E-value of 8e-147, indicate that the aquaporin-1 protein from the Canis lupus familiaris, or dog family, has a very similar sequence to the bovine AQP1. The similar protein sequence is a good indicator that the two proteins have similar function. Research has shown that the AQP1 cDNA in dog kidney is homologous to cDNA of bovine, human, mouse and rat origin. The cDNA in bovines was found to be the closest to that of dogs among the species reported. Both the dog AQP1 and the bovine AQP1 was constructed with 271 amino acids. The amino acid sequence homology between dog and bovine was very high at 94%. However, some LP (loop) regions in these animals AQP1 showed remarkable substitutions with different amino acids. For example, LP1 showed considerable variation between these species: 38% of amino acids in the peptide sequence of the LP 1 in dog AQP1 were different from those in bovines. Furthermore, two additional hydrophobic amino acids were added in the LP1 of dog AQP1. Therefore, the LP1 in dog AQP1 might be rather hydrophobic compared with other species(5).
Structural studies have revealed that all AQPs share the same basic architecture, which consists of two tandem repeats, each containing a bundle of three transmembrane alpha-helices and a hydrophobic loop with the highly conserved asparagine, proline, alanine (NPA) motif(2). A Z score of 35.2 for the sheep AQP0 protein (PDB ID=2B6O) from the Dali Server indicates that AQP0 has a similar tertiary structure to AQP1(3). In contrast to the AQP1 protein, the AQP0 protein is normally found in lens fiber cell membranes of the eyes. However, it is still responsible for moving water across a membrane although at rates substantially lower than for AQP1 under similar conditions. The pores of the AQP1 channel are critically narrow and chemically optimized to facilitate specific but rapid water transport. In the case of AQP0, a small number of bulky, typically aromatic, residues take the place of what in AQP1 are smaller pore-lining residues. In their lowest energy configurations, these bulky residues block water access to the pore as seen in both the EM- and X-ray-based structures of AQP0(4). This shows that often times protein primary and tertiary structures are conserved; a slight structural modification can have a large impact on the protein's function.
The functional unit of AQP1 is a tetramer with each monomer providing an independent water pore (6). The Phi/Psi angles for AQP1 are shown in Image 1. The predicted monomer size of the mammalian aquaporins ranges from 26 to 34 kDa (7). Each monomer contains six transmembrane helices packed to form part of a trapezoid-like structure when viewed normal to the membrane plane. Two membrane-inserted but non-membrane-spanning helices, which define a major portion of the pore, are partially enclosed by this structure and form an integral part of the outer wall (8). The polypeptide contains an internal repeat, the N- and the C-terminal halves are sequence related, and each contains the signature motif Asn-Pro-Ala. Each sequence half contains an NPA (asparagine, proline, alanine) motif, which is conserved throughout the aquaporin superfamily (1). The conservation of arginine, histidine and phenylalanine side chains at their respective locations within the constriction region in the known water channels is a strong indicator of channel water specificity (6).
The AQP1 monomers organize as tetramers. Within the lipid bilayer, the buried surface at the monomer interfaces is mostly hydrophobic. This surface is outlined by residues from transmembrane alpha-helix 1 (TM1), TM2 and TM4, TM5 belonging to adjacent monomers that form a tightly packed, traditional, left-handed helix bundle (9). Between the adjacent monomers, the side chain of T-158, in the TM4-TM5 linker, hydrogen bonds with side chains of Q-67 and S-68 at the cytoplasmic surface, whereas on the extracellular face, the side chain of Y188 hydrogen bonds with Q-43 (in the TM1-TM2 linker). Thus, the stability of the tetramer is derived both from hydrophobic interactions within and polar interactions outside the bilayer (10).
Half of the channel wall along the selectivity filter can be considered hydrophobic and the other half hydrophilic. The hydrophilic face provides the chemical groups that are essential for displacing certain waters of hydration and therefore establishes a pathway for coordinating water transport (1). In AQP1, sufficient hydrogen-bond-forming groups are available so that water molecules can readily move through the constriction region. These hydrogen-bond-forming groups are provided by constriction region residues H-182, which is conserved across the water-specific aquaporins and R-197, which is conserved throughout most of the aquaporin superfamily, and the backbone carbonyl oxygens of residues G-190, C-191 and G-192 (6). Water molecules have been identified at four locations within the AQP1 selectivity filter. The first water molecule is located adjacent to the constriction region about 7 Å from the pseudo two-fold axis towards the extracellular surface. At this location the water is coordinated by hydrogen bonds established with the 2 nitrogen of H-182 and the backbone carbonyl oxygen of G-192. The next two waters are centered about the pseudo two-fold axis, one hydrogen-bonded to the 2 nitrogen of N-194 and the other hydrogen-bonded to nitrogen 2 of N-78. The fourth water visible in the channel is located near the cytoplasmic end of the selectivity filter. The backbone carbonyl oxygens of residues H-76 and A-75 form the coordinating hydrogen bonds with this water. These four waters do not form a contiguous hydrogen-bonded chain as only the middle two are close enough to each other to form a water-water hydrogen bond (6). In AQP1 the residue most critical in supporting rapid water throughput, while hindering the passage of glycerol, is H-182. Throughout the aquaporin family the choice of amino acid at this location appears to be essential in defining whether an aquaporin will be specific for water or additionally selective for other solutes such as glycerol (6).