Structural and functional studies of aquaporins

Aquaporin (AQP) is a family of membrane channel proteins that specifically transport water and/or small solutes. AQP homologs have been found in nearly all forms of life including archaea, eubacteria, fungi, plants and animals. So far, thirteen mammalian AQP genes have been reported and characterized. They are widely expressed in numerous tissues/organs. According to the protein sequence alignment and different permeabilities, AQPs are further classified into two subgroups. The aquaporin subfamily is only permeable to water, and the aquaglyceroporin subfamily transports small solutes (such as glycerol and urea) as well as water.; AQP6 belongs to the aquaporin subfamily. Unlike other AQPs, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. Amino acid sequence alignment and modeled structure both indicated that there is a unique asparagine residue (N60) in transmembrane domain (TM) 2 of AQP6. To test the hypothesis that N60 introduces instability to the TM2-TM5 interaction and that the instability may contribute the rapid gating of the anion permeability of AQP6, site-directed mutagenesis was performed. The asparagine residue was replaced by glycine, alanine or serine and the water or anion permeability of each mutant (N60G, N60A or N60S) was determined by the oocyte swelling assay and electrophysiological recordings. Our results showed that a single residue substitution (N60G) converted AQP6 from an anion channel to a water channel. This suggests that N60 may act as a teeter board needed for rapid structural oscillations between TM2 and TM5 during anion permeation.; AQP8 is another member of water-selective aquaporin subfamily. It is widely expressed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonia, but no direct characterization had yet been reported. For direct functional characterization, recombinant rat, human and mouse AQP8 proteins were purified from yeast and reconstituted into proteoliposomes. Using a stopped-flow spectrophotometer, all three AQP8 proteoliposome samples exhibited significant water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. Rat and human AQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. Human AQP8-injected oocytes had significantly higher [14C]methylammonium uptake than control oocytes. Since formamide and methylammonium are used as ammonia analogues, our results suggest that AQP8 may transport ammonia in vivo and physiologically contribute to the acid-base equilibrium.; AQP7, a member of the aquaglyceroporin subfamily, is permeable to water, glycerol, urea and arsenite. It is expressed in adipose tissue, kidney and testis. Recent studies in human subjects and AQP7 null mice have shown that AQP7 is involved in fat metabolism. To determine whether AQP7 is a risk factor for human health, AQP7 SNPs (single nucleotide polymorphisms) were screened among 2071 individuals. Twelve non-synonymous SNPs in the coding region of AQP7 were found by searching two databases. Three of them, E40K, N94S and G264V, seemed important for AQP7 structure and/or function through sequence alignment and structural modeling. In functional characterization, N94S transported water and glycerol similar to wild-type AQP7. In contrast, E40K and G264V, did not transport water or glycerol and had much lower expression on the oocyte plasma membrane than wild-type AQP7. In the SNP-screening, no individuals carrying E40K or N90S were found. However, there were some G264V heterozygotes. The G264V SNP was dependent on ethnicity. There were only 0.5% of African-Americans and 0.6% of Ghanaians carrying this SNP, whereas 8.9% of Caucasians, 9.3% of European centenarians and 10.3% of normal Europeans had this SNP. The frequency of G26...