In the colony of Sprague-Dawley (SD) strain, we found that there were rats expressing a mutant AQP5, which has a point mutation at nt 308 (G308A), leading to a replacement of 103Gly with 103Asp in the 3rd transmembrane domain. The mutant molecule scarcely expressed in the acinar cells, probably because of ineffective trafficking. The mutant molecule, however, showed normal water permeability when assessed by the oocyte system

Akamatsu, Tetsuya

Azlina, Ahmad

Hasegawa, Takahiro

Hosoi, Kazuo

Karabasil, Mileva R

Murdiastuti, Kwartarini

Purevjav, Javkhlan

Purwanti, Nunuk

Yao, Chenjuan

Tokushima University Institutional Repository

INTRODUCTIONAQP is a channel protein expressed in virtuallyall living cells. There are 13 members in mammalsand they are generally responsible for rapid watermovement across the plasma membrane in almostall cells (1, 2). AQP5, a member of this family pro-teins is expressed in the apical membrane of mul-tiple secretory glands, including the lacrimal, sali-vary, and airway submucosal glands, type 1 alveo-lar cells (3, 4), sweat glands (5), corneal epithelium(6), and duodenal Brunner’s gland (7). We foundthat the expression level of the AQP5 protein in thesubmandibular glands (SMG) was divergent amongindividual SD rats, and identified a point mutationin AQP5 gene of rats expressing AQP5 protein atlow level. In the present study, we characterized thismutant AQP5 with respect to its ability to affordwater permeability and to undergo membrane traf-ficking/translocation.METHODSWater permeability of wild type and mutant AQP5was determined by Zenopus oocytes osmotic assay.Western blotting, RT-PCR/real-time PCR were em-ployed for analysis of AQP5 proteins and its mRNA.Trafficking of AQP5 protein was measured undera confocal laser scanning microscope by using theMDCKII cells transiently expressed with GFP-AQP5.PROCEEDINGEffects of natural point mutation of rat aquaporin 5 ex-pressed in vitro on its capacity of water permeabilityand membrane traffickingMileva R Karabasil1, 2 , Kwartarini Murdiastuti3 , Nunuk Purwanti1,4, Ahmad Azlina1,Purevjav Javkhlan1,5, Takahiro Hasegawa1, Chenjuan Yao1, Tetsuya Akamatsu1, andKazuo Hosoi11Department of Molecular Oral Physiology, Institute of Health Biosciences, the University of TokushimaGraduate School, Tokushima, Japan ; 2Department of Orthodontics, Faculty of Dentistry, BelgradeUniversity, Belgrade, Serbia ; 3Department of Periodontology, 4Deapartment of Basic Dental Science,Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia ; and 5Department of Periodon-tology and Endodontology, Institute of Health Biosciences, the University of Tokushima GraduateSchool, Tokushima, JapanAbstract : In the colony of Sprague-Dawley (SD) strain, we found that there were rats ex-pressing a mutant AQP5, which has a point mutation at nt 308 (G308A), leading to a re-placement of 103Gly with 103Asp in the 3rd transmembrane domain. The mutant moleculescarcely expressed in the acinar cells, probably because of ineffective trafficking. The mu-tant molecule, however, showed normal water permeability when assessed by the oocytesystem. J. Med. Invest. 56 Suppl. : 398-400, December, 2009Keywords : AQP5 mutant, trafficking, salivary glandReceived for publication November 18, 2009 ; accepted Novem-ber 25, 2009.Both authors contributed equally to this workAddress correspondence and reprint requests to Kazuo Hosoi,Department of Molecular Oral Physiology, Institute of HealthBiosciences, the University of Tokushima Graduate School,Kuramoto-cho, Tokushima 770-8504, Japan and Fax : +81-88-633-7324.The Journal of Medical Investigation Vol. 56 Supplement 2009398RESULTS AND DISCUSSIONA greater than 2-fold diversity in the expressionlevel of aquaporin 5 (AQP5) protein has been ob-served in the membrane fraction of the SMG in SDrats (8). Breeding between brother and sister ratswas repeated within high AQP5-producers and lowones to obtain inbred offspring. By Western blotting,levels of AQP5 protein in the parotid and lacrimalglands, and lungs were all low in low producers,whereas they were all high in high producers, im-plying genetic variations of the gene for this waterchannel. Despite this implication, AQP5 mRNA lev-els were almost the same between the 2 groups byNorthern blotting and real-time RT-PCR, suggest-ing the irrelevance of transcriptional regulation forthis diversity. AQP5 cDNAs from the SMGs of the2 groups were sequenced. The nucleotide sequenceof AQP5 cDNA from low producers indicated theexistence of a point mutation at nt 308 (G308A),leading to a replacement of 103Gly with 103Asp in the3rd transmembrane domain (Fig. 1) ; but no altera-tion was detected in the Kozak area (9). The exis-tence of such a mutation was confirmed by the as-sessment of genomic DNA also. The mutant AQP5expressed in Xenopus oocytes showed water perme-ability similar to those expressed by the normalmolecule. The mutant and wild-type GFP-AQP5’sFig. 1 Location of a point mutation in the AQP5 cDNA and the deduced amino acid sequence as compared with human AQP1.Point mutation is located in the 3rd transmembrane domain, where glycine is replaced with aspartic acid. Point mutation is locatedat the remote site from the aqueous pore in the membrane, implying that mutation may not affect the AQP5 function. From Murdiastuti,et al .(9)Location of the point mutation in rat AQP5 moleculeAmino acid residue facing to the inside of the aqueous pore in the membrane From Murata, et al . (10).Amino acid residues of the 3rd transmembrane domain, which are located at the remote site from the aqueous pore in the mem-brane.Underlines, the NPA motif conserved throughout the family. Bold letter, trans membrane domains.The Journal of Medical Investigation Vol. 56 Supplement December 2009 399expressed in MDCK-II cells stayed in the cytoplas-mic compartment by 12 h, and were then translo-cated to the apical plasma membrane at 24 and 48 h.During translocation, involvement of microtubules,but not phosphorylation of AQP5 at Ser/Thr PKAtarget motif (152SRRTS) were suggested. At 24 and48 h, the apical localization of mutant GFP-AQP5was less than that of the wild-type molecule. Thap-sigargin, an inhibitor of ER Ca2+-ATPase, inducedthe rapid trafficking of AQP5 ; and the mutant mole-cule showed significantly reduced membrane traf-ficking comparing to the wild-type molecule (11).In frozen sections of the SMG from mutant rats,but not in those of the wild-type gland, a relativelylarge number of AQP5-positive structures appearedin the cytoplasm of the acinar cells, which structureswere also immuno-positive for LAMP2, a lysosome-associated membrane protein, suggesting that mostof the mutant AQP5 molecule entered lysosomesfor degradation.REFERENCES1. Verkman AS, Mitra AK : Structure and functionof aquaporin water channels. Am J Physiol Re-nal Physiol 278 : F13-F28, 20002. Verkman AS : More than just water channels :unexpected cellular roles of aquaporins. J CellSci 118 : 3225-3232, 20053. Raina S, Preston GM, Guggino WB, Agre P :Molecular cloning and characterization of anaquaporin cDNA from salivary, lacrimal, andrespiratory tissues. J Biol Chem 270 : 1908-1912, 19954. Nielsen S, King LS, Christensen BM, Agre P :Aquaporins in complex tissues. II. Subcellulardistribution in respiratory and glandular tis-sues of rat. Am J Physiol Cell Physiol 273 :C1549-C1561, 19975. Nejsum LN, Kwon TH, Jensen UB, FumagalliO, Frokiaer J, Krane CM, Menon AG, King LS,Agre PC, Nielsen S : Functional requirementof aquaporin-5 in plasma membranes of sweatglands. Proc Natl Acad Sci USA 99 : 511-516,20026. Hamann S, Zeuthen T, La Cour M, NagelhusEA, Ottersen OP, Agre P, Nielsen S : Aquaporinsin complex tissues : distribution of aquaporins1-5 in human and rat eye. Am J Physiol CellPhysiol 274 : C1332-C1345, 19987. Parvin MN, Kurabuchi S, Murdiastuti K, Yao C,Kosugi-Tanaka C, Akamatsu T, Kanamori N,Hosoi K : Subcellular redistribution of AQP5by vasoactive intestinal polypeptide in theBrunner’s gland of the rat duodenum. Am JPhysiol Gastrointest Liver Physiol 288 : G1283-G1291, 20058. Murdiastuti, K, Miki O, Yao C, Parvin MN,Kosugi-Tanaka C, Akamatsu T, Kanamori N,Hoso, K : Divergent expression and localizationof aquaporin 5, an exocrine-type water channel,in the submandibular gland of Sprague-Dawleyrats. Pflügers Arch Eur J Physiol 445 : 405-412,20029. Murdiastuti K, Purwanti N, Karabasil MR, LiX, Yao C, Akamatsu T, Kanamori N, Hosoi K :A naturally occurring point mutation in the rataquaporin 5 gene, influencing its protein pro-duction by and secretion of water from salivaryglands Am J Physiol Gastrointest Liver Physiol291 : G1081-G1088, 200610. Murat K, Mitsuoka K, Hirai T, Walz T, Agre P,Heymann JB, Engel A, Fujiyoshi, Y : Struc-tural determinants of water permeation throughaquaporin-1. Nature 405 : 599-605, 200011. Karabasil, MR : A naturally occurring rat AQP5G103D mutant exhibits normal water perme-ability but reduced membrane trafficking andinfluence the tight junction-protein expression.Dissertation submitted to The Graduate Schoolof Oral Sciences the University of Tokushima2009M. R. Karabasil, et al. Characterization of rat mutant AQP5400

Effects of natural point mutation of rat aquaporin 5 expressed in vitro on its capacity of water permeability and membrane trafficking

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Effects of natural point mutation of rat aquaporin 5 expressed in vitro on its capacity of water permeability and membrane trafficking

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