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First published online November 4, 2005
Journal of Experimental Biology 208, 4203-4211 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01868

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Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming

Masatomi Hosoi^*, Kazuharu Takeuchi, Hideki Sawada and Haruhiko Toyohara

Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

View larger version (84K): [in a new window]   Fig. 1. Amino acid sequence alignment of mussel taurine transporter (muTAUT) with fish and mammalian taurine transporters. Dots indicate amino acids identical to those of muTAUT. Hyphens indicate gaps. Gray boxes indicate the 12 putative membrane-spanning domains. Asterisks in the second extracellular domain show the potential N-glycosylation sites of muTAUT. Accession numbers of each sequence are as follows, carp: AB006986, tilapia: AB033497, dog: M95495, and human: U09220.

View larger version (16K): [in a new window]   Fig. 2. Kinetics of taurine uptake into Xenopus oocytes injected with mussel taurine transporter (muTAUT) synthetic RNA. Uptake of taurine into cRNA-injected oocytes was measured at indicated taurine concentrations with 0.5 µCi ml-1 [3H]taurine. Values obtained under the same conditions with uninjected oocytes were subtracted from values of corresponding injected samples. An Eadie-Hofstee plot of the data is depicted in the inset.

View larger version (20K): [in a new window]   Fig. 3. Dependence of taurine uptake on [Na+] and [Cl-] in mussel taurine transporter (muTAUT)-injected oocytes. Taurine uptake was measured at (A) various Na+ (1-250 mmol l-1) concentrations and constant Cl- concentration (250 mmol l-1) or (B) various Cl- (1-250 mmol l-1) concentrations and constant Na+ concentration (250 mmol l-1), with 10 µmol l-1 taurine containing 0.5 µCi ml-1 [3H]taurine. Isotonicity of the medium was maintained with choline chloride or sodium gluconate. Plotted data were adjusted by subtraction of uptake values by water-injected oocytes from those by muTAUT-injected oocytes (N=6-8). Error bars represent the standard deviation. The Hill coefficient and K50 were estimated by linear fitting of Hill plots (insets).

View larger version (19K): [in a new window]   Fig. 4. Competition analysis of taurine uptake by mussel taurine transporter (muTAUT)-injected oocytes. Taurine uptake in muTAUT- and water-injected oocytes was measured with 10 µmol l-1 taurine containing 0.5 µCi ml-1 [3H]taurine in the presence of 1 mmol l-1 unlabeled compounds as the competitor. Each column represents the mean ± standard deviation of 5-8 oocytes.

View larger version (25K): [in a new window]   Fig. 5. Relationship of taurine uptake and NaCl concentration/medium osmolality in mussel taurine transporter (muTAUT)- and tilapia taurine transporter (tTAUT)-injected oocytes. Closed symbols indicate taurine uptake in various NaCl concentrations indicated in the lower horizontal axis. Open symbols represent taurine uptake in 100 mmol l-1 NaCl concentration with various medium osmolalities adjusted by the addition of glycerol (upper horizontal axis). Values obtained under the same conditions with uninjected oocytes were subtracted from those of corresponding injected samples.

View larger version (38K): [in a new window]   Fig. 6. Northern blot analysis of taurine transporter mRNA in mussels exposed to changes in ambient salinity. (A,B) Time course of mussel taurine transporter (muTAUT) mRNA abundance in the mantle, gill and adductor muscle of mussels exposed to 2x seawater (A), 0.5x seawater (B), respectively. Total RNAs extracted from three mussels and mixed for minimization of individual variability. Lower panels show the rRNA signals of ethidium bromide-stained gel. (C) Depression of muTAUT mRNA expression by the addition of taurine. Northern blot analysis was performed using 20 µg of total RNAs from the mantle of mussels exposed to 0.5x seawater with or without 25 mmol l-1 taurine for 24 h. RNAs loaded in each lane were extracted from different specimens.

View larger version (171K): [in a new window]   Fig. 7. Immunohistochemical detection of taurine transporter on the mantle of mussels exposed to changes in ambient salinity. (A,B) Mantle sections and (C,D) gill sections of mussels acclimated to 1x seawater (A,C) or exposed to 0.5x seawater for 48 h (B,D). The brownish color on the surface of the mantle (indicated by arrows in A) is not staining but endogenous pigments. Scale bars, 50 µm.