title: Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism.
abstract: The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.
authors.affiliation: Department of Medicinal and Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: von Bülow
authors.forename: Annika
authors.initials: A
authors.lastname: Müller-Lucks
authors.forename: Lei
authors.initials: L
authors.lastname: Kai
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.forename: Eric
authors.initials: E
authors.lastname: Beitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014867:Water / Q000737:chemistry / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D020346:Aquaporins; D015800:Protozoan Proteins; D014867:Water