Molecular Cloning and Characterization of an Insect Aquaporin

Abstract

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, F., Le Cahérec, F., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J.F., Gouranton, J. & Thomas, D. (1995) J. Biol. Chem. 270, 17414-17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin 1, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert, J.F. (1996) J. Cell Sci. 109, 1285-1295]. In the present work, we cloned a full-length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26-kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein gamma, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15-fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrhenius activation energy of 11.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aquaporin 1. In the Xenopus oocyte system, Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions.

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

“…Aquaporins have been divided into two types: one subgroup is highly selective for the passage of water, and the other subgroup transports water as well as small neutral solutes such as glycerol. Experimental studies on the transport properties are facilitated by the fact that the water-conducting properties of these channels are inhibited by mercuric chloride, HgCl 2 (Preston et al, 1992), as also shown for an insect aquaporin (Le Caherec et al 1996b). This inhibition apparently is due to binding of the mercury molecule with the aquaporin protein in a narrow part of the pore, so occluding the aqueous pathway (Murata et al 2000).…”

The Current Knowledge of Invertebrate Aquaporin Water Channels with Particular Emphasis on Insect AQPs

“…Research of aquaporins in Cicadella viridis began with the filter chamber [18], a structure, built by highly specialized epithelial cells, localized in the digestive tract. It is responsible for the rapid transport of water from the initial midgut, to the terminal midgut and Malpighian tubules.…”

“…Because these cysteines are localized near the transmembrane domains, access to them by ions is difficult. Therefore, a higher concentration of ions is most likely necessary to inhibit water permeability [18] (Fig. 3).…”

“…Firstly, its amino acid sequence is 43% identical to AQP1 [7]. The rate of the water transport through AQPcic is faster than through Rp-MIP, but is not as fast as through AQP1 [18]. Water permeability can be inhibited by mercury ions, but the cause of this phenomenon is unclear.…”

“…mRNA isolated from the epithelial cells of the filter chamber encodes an aquaporin, consisting of 255 amino acids (26 kDa), which has been named AQPcic. mRNA for this protein was injected into X. laevis oocytes to determine the kind of solute that can be transported through this water channel [18]. In comparison with the control, water transport through the oocyte membrane with injected mRNA for the aquaporin, increased 15-fold.…”