title: Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion.
abstract: 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.
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Wree
authors.forename: Binghua
authors.initials: B
authors.lastname: Wu
authors.forename: Thomas
authors.initials: T
authors.lastname: Zeuthen
authors.forename: Eric
authors.initials: E
authors.lastname: Beitz
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000818:Animals; D051397:Aquaglyceroporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020346:Aquaporins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry*; D015153:Blotting, Western; D057508:Burkholderia cenocepacia / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D004594:Electrophysiology; D005990:Glycerol / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism; D014981:Xenopus / Q000235:genetics / Q000378:metabolism
chemical_list: D051397:Aquaglyceroporins; D020346:Aquaporins; D002412:Cations; D008744:Methylamines; D014867:Water; D001216:Asparagine; C027451:methylamine; D005990:Glycerol; D011188:Potassium