The Role Of NPA Motifs In Membrane Targeting Of AQP4

The aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins as special water-transporting channels that are expressed widely in prokaryotic cell and eukaryotic cells, with 13 members identified to date in mammals. A lot of studies about AQPs gene mutation and knockout mice showed that AQPs play an important role in water transductions of physiological conditions. Recent studies indicated that AQPs play a role in the physiological processes of tumor angiogenesis,cell migration and apoptosis. The most abundant water channel in the CNS is AQP4, which is localized to ependyma cells and the endfeet of astrocytes in contact with the blood vessels of the blood–brain barrier and in astrocytic processes in contact with synapses. The studies of AQP4 knockout mice showed AQP4 is primarily responsible for cerebral water homeostasis.With the deep investigation of the functions of AQPs, the relationship between specific motif of gene sequence and the biogenesis became a hot region. In most cell types the AQPs reside constitutively at the cell plasma membrane, with the notable exception of AQP2 in kidney collecting duct where vasopressin regulates AQP2 trafficking between endosomes and the cell plasma membrane. AQP11 and AQP12 also can not express in cell membrane. Although the specific gene sequence of each AQPs water channel (30% homology), almost each member of AQPs has two highly conserved NPA motifs (asparagine-proline-alanine, NPA motifs). Crystallographic studies of some AQPs demonstrated that the two NPA motifs are localized in the narrow central constriction of the channel, where they play a crucial role in water permeation. Few AQP members with variations in NPA motifs such as AQP11 and AQP12, in which the second NPA motif was changed by NPC and NPT, do not express in the plasma membrane , suggesting an important role of NPA motifs in AQP plasma membrane targeting.AQP4 has two alternative splice variants resulting from differential translation initiation either at the first methionine (AQP4-M1, 323 aa) or at the second methionine (AQP4-M23, 301 aa). As a result, AQP4-M1 has a 22 residues longer N-terminus than AQP4-M23. High- resolution crystal structures of AQP4 showed that the asparagine of each NPA motif forms hydrogen bonds to a water molecule in the center of the channel, which is different from AQP1 where two asparagines from both NPA motifs form hydrogen bonds to one water molecule. However, role of NPA motifs in AQP plasma membrane targeting remains largely unknown.In this study, we examined the role of NPA motifs in membrane targeting and biogenesis of AQP4, and whether there is some difference between AQP4-M1 and AQP4-M23 or not. In our experiments, we examined the role of NPA motif in the biogenesis of AQP4-M1 and AQP4-M23 by mutagenesis and cell transfection. In order to know whether NPA motifs play a role or not in biogenesis of AQP4 and the difference in biogenesis of AQP4-M1 and AQP4-M23, we constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in M1 and M23 isoforms of AQP4 and analyzed their expression patterns in transfected cell models. The result suggests that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs interact with other structural domains in the regulation of plasma membrane targeting of AQP4.