Identification of a new water channel (Rg-MIP) in the Malpighian tubules of the insect Rhodnius prolixus

Malpighian tubules (MT) of Rhodnius prolixus transport fluid at very high r ates. To identify whether aquaporins (AQPs) are present in the MT of X. pro lixus, total ribonucleic acid (RNA) was isolated from MT and used in a reve rse transcription, polymerase chain reaction (RT-PCR), with two degenerate primers to highly conserved regions of the members of the AQPs family. A de oxyribonucleic acid (DNA) fragment of 370 bp was amplified; its sequence re vealed a novel protein, representing a new member of the major intrinsic pr otein (MIP) family. The complementary DNA (cDNA) sequence of this new MIP p rotein was cloned by using RNA from MT and the rapid amplification of cDNA ends (RACE) technique. The cDNA had 1133 bp and the largest open leading fr ame coded for a protein of 286 amino acids, named R. prolixus major intrins ic protein (Rp-MIP). The hydrophobicity profile of the amino acid sequence predicts six transmembrane domains. Northern blot analysis of MT RNA showed a single transcript of about 1-1.3 kb for Rp-MIP RT-PCR of single isolated MT and in situ hybridization analysis showed Rp-MIP transcripts in both pr oximal and distal segments. Expression of Rp-MIP in Xenopus laevis oocytes doubled the osmotic water permeability P-f, indicating that Rp-MIP may func tion as an aquaporin protein in the MT of the insect and thus may participa te in urine formation in R. prolixus.