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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. | 1FEBS Journal Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion Dorothea Wree1 Binghua Wu1 Thomas Zeuthen2 and Eric Beitz1 1 Department of Pharmaceuticaland MedicinalChemistry Christian-Albrechts-University of Kiel Germany 2 Institute of Cellular and Molecular Medicine University of Copenhagen Denmark Keywords aquaglyceroporin aquaporin potassium proton sodium Correspondence E. Beitz PharmaceuticalInstitute Christian-Albrechts-University of Kiel Gutenbergstrasse 76 24118 Kiel Germany Fax 49 431 880 1352 Tel 49 431 880 1809 E-mail ebeitz@pharmazie.uni-kiel.de Website http www.pharmazie.uni-kiel.de chem Received 8 November 2010 revised 10 December 2010 accepted 13 December 2010 doi 10.1111 j.1742-4658.2010.07993.x 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 .
