Importance of NPA motifs in the expression and function of water channel aquaporin-1

Abstract

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic
studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving
to bind water molecules for selective and efficient water passage. To investigate the importance of the two NPA motifs in
the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 deletion
and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector
pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence
indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO
cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional
analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2
double deletion had little effect on 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 AQP1 water
channel.