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Two full length AQP genes (LrAQP-A and LrAQP-G) were isolated from the earthworm Lumbricus rubellus by RACE-PCR. Sequence analysis revealed a number of unusual residues in the critical aqueous pore region in both AQPs, including a substitution in the first canonical NPA box from an alanine residue to a glycine in LrAQP-G. This unusual NPG motif was also identified in AQP genes isolated from a further 6 annelids studied suggesting that although the NPG motif is unique amongst Animalia studied to date, it is common within the annelids. In addition to this substitution, LrAQP-G contains an insertion of 10 amino acids in its first extracellular loop and putative phosphorylation sites in the intracellular C terminus. LrAQP-A also contained unusual pore substitutions and putative phosphorylation sites at the C terminus. Phylogenetic analysis suggests LrAQP-A is more closely related to other invertebrate AQPs whereas LrAQP-G clusters on a branch of its own. Both AQPs were present in all tissues examined, including the major osmoregulatory tissues; integument, nephridia and gut. qPCR revealed that LrAQP-A transcripts are more abundant than LrAQP-G in the integument, reproductive organs, calciferous glands and nephridia. In situ hybridisation revealed both transcripts to be largely co-localised in discrete cell types in the outer epidermal cells of the bodywall, the coleomic surface of the gut and in the endothelia of the nephridia. Functional characterisation in oocyte swelling assays revealed that LrAQP-A facilitates the mercury-sensitive transport of water at similar rates to human AQP-5. The rate of water transport was increased significantly by lowering the pH of bathing media. LrAQP-G also facilitated water transport but at rates ~50% less than LrAQP-A and was mercury and pH insensitive. Neither AQP transported glycerol or urea but interestingly LrAQP-A is permeable to NH3 in a pH-dependent manner. Both LrAQP-A and LrAQP-G showed partial loss of function after PKC-mediated phosphorylation. In environmental stress experiments the expression of both genes was modulated significantly. In worms exposed to wet soil both the integument and nephridia gene expression of LrAQP-A was reduced. In hyposmotic saline, LrAQP-G was downregulated in the integument. In hyperosmotic saline LrAQP-G was again downregulated in the integument but was upregulated in the gut. LrAQP-A and LrAQP-G are intimately involved with water transport in L. rubellus. In addition they seem to be modulated in the short and long term dependent on osmotic conditions. |
