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Background information Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208–219). Results We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf = 69 μm/s) to the same extent as AqpB Δ208–219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208–219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 μm/min vs. 6.3 μm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. Conclusion Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219).We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf = 69 μm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 μm/min vs. 6.3 μm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established.Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

Infectious diseases, caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a global threat. The increase in the number of affected individuals and the rapid spread of drug-resistant strains call for specific novel strategies to combat human pathogenic parasites. In the search for novel drug targets, transport proteins for nutrients and metabolites of the parasite—host interface are getting into focus. The present review summarizes and discusses the currently available results on protozoan aquaporins. Various genes coding for aquaporin water and solute channels have been identified in the protozoan genomes and they are probable elements of the parasite's cell membrane. Phylogenetic analysis reveals that individual aquaporin genes are of bacterial or plant origin. So far, six protozoan aquaporins have been cloned and functionally characterized. Typically, these are bifunctional channels and pass water at intermediate to high rates as well as uncharged solutes. In the present review, amino acid compositions of the individual pore entries are compared and permeability properties are attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism are discussed. Finally, the potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds is reviewed.

Background information. Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. Results. We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP—AQP6 or HA—AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin—Darby canine kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6–GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. Conclusion. Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.