Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats.
BACKGROUND
AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals.


RESULTS
Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation.


CONCLUSIONS
Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.
Department of Molecular Oral Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima-shi, Tokushima 770-8504, Japan.
Mileva Ratko
MR
Karabasil
Takahiro
T
Hasegawa
Ahmad
A
Azlina
Nunuk
N
Purwanti
Chenjuan
C
Yao
Tetsuya
T
Akamatsu
Shigemasa
S
Tomioka
Kazuo
K
Hosoi
D000595:Amino Acid Sequence; D000818:Animals; D051402:Aquaporin 5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010539:Permeability; D017354:Point Mutation; D021381:Protein Transport; D051381:Rats; D016415:Sequence Alignment; D013363:Submandibular Gland / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000378:metabolism*; D014981:Xenopus
D051402:Aquaporin 5; D014867:Water