[
Biochim Biophys Acta,
2000]
A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.
[
International Worm Meeting,
2003]
Water movement across the cell membrane is a fundamental process for the maintenance of water homeostasis in many cell types. The aquaporin (AQP) water channels belong to the major intrinsic protein (MIP) superfamily and play a central role in water transport. AQPs are present in many forms of life including bacteria, yeast, plants, insects, and mammals. There are at least eleven AQPs (AQP0-AQP10) in mammals which functionally characterized in terms of the permeability of water and small solutes. The primary structures of AQPs are almost similar in size (250-290 amino acids). Six membrane-spanning domains and two highly conserved amino acid residues called NPA (asparagine-proline-alanine) boxes are the common features of AQP. The completion of genome sequence of the nematode Caenorhabditis elegans revealed the presence of eight MIPs in its genome. Of these MIPs, AQP-CE1 (C01G6.5) and AQP-CE2 (F40F9.9) have been characterized as water permeable channels when expressed in Xenopus oocytes. AQP-CE1 and CE2 correspond to AQP3 and AQP8 in mammals, respectively, from sequence homology and channel characteristics. The remaining "typical" MIPs in C. elegans are also thought to function as water channels, although physiological roles are still unknown. We have recently identified and characterized the novel type of aquaporin, AQP11 and AQP12 in mouse, which did not permeate water. We found two homologs in C. elegans genome, ZK525.2 (III) and ZK1321.3 (II). ZK525.2 encoded 290 amino acids and ZK1321.3 encoded 280 amino acids having homology with AQP11. When cRNAs were expressed in Xenopus oocyte, they did not permeate water as AQP11. To understand the physiological roles of new AQP genes in C. elegans, we investigated expression patterns and knock out phenotypes of ZK525.2 and ZK1321.3. Expression pattern of ZK525.2::GFP and ZK1321.3::GFP translational fusion constructs were almost similar; both were strongly expressed in intestine, and weakly in other minor tissues. RNAi of ZK525.2 or ZK1321.3 resulted in no obvious phenotype when it was done solely. Since AQP11 was expressed in small intestine in mouse, ZK525.2 and ZK1321.3 products may have redundant roles in C. elegans intestine. We are now trying to observe clear phenotype by double RNAi. We will also present the progress on expression and RNAi analysis for typical MIPs in C. elegans.