Expression and functional characterization of NPA motif-null aquaporin-1 mutations.
ABSTRACT The asparagine-proline-alanine sequences (NPA motifs) in Loops B and E of aquaporin are highly conserved. To investigate the role of two NPA motifs in the structure and function of aquaporin water channels, we generated human aquaporins (AQP)-1 mutations with NPA1 deletion, NPA2 deletion and NPA1,2 double deletion. Immunoblotting and immunofluorescence analysis indicated that all the three human AQP1 mutants possessed identical protein pattern and similar plasma membrane expression pattern compared to wild-type AQP1. Plasma membrane osmotic water permeability analysis, measured by YFP-based fluorescence quenching method and Xenopus oocyte expression assays, demonstrated that NPA1 or NPA2 deletion significantly reduced human AQP1 water permeability nearly 50% compared to wild-type AQP1, while NPA1,2 double deletion had little effect on human AQP1 water permeability. These results provide evidence that NPA motifs are important for water permeation but not essential for the expression, intracellular processing and the basic structure of human aquaporin 1.