Regulation und gating von neuen Aquaporinen aus dem Schleimpilz Dictyostelium disciodeum und deren Funktion für die Zellmotilität

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We have characterized a novel aquaporin, AqpB, from Dictyostelium discoideum. AqpB is water-specific, O-glycosylated and exhibits and a novel gating mechanism. It is localized in vacuolar structures, the plasma membrane, and in membrane protrusions. So far, it represents the sole functional aquaporin in Dictyostelia und is expressed in all developmental stages. The critical domain for the gating comprises seven amino acid positions in the D-loop. Until now, knockout attempts have yielded unspecific genome integration. Besides AqpB, we have identified a second aquaporin, AqpC, in amoebae on the cDNA and protein level. Now we will further elucidate the gating mechanism of AqpB and analyze the physiological function mainly of AqpB and more initially of AqpC. We will generate new mutations of AqpB especially in the sequence stretch around the conserved tyrosine-218 for biophysical analysis. We will construct new knockout plasmids with longer linker arms and select GC-rich sequences within the coding region of AqpB to improve the specificity of genomic integration. If the knockout is lethal, we will apply the amber-suppression knockdown method, which reduces protein levels up to 90%. By using dominant overexpression of de-regulated AqpB-Variants, we will test for the role of gating with respect to cell physiology. We will analyze the phenotypes of the knockout/knockdown/overexpression cultures regarding morphology, cytokinesis, development, motility, volume regulation, and macropinocytosis. We expect to gain basic knowledge on the role of aquaporins in cell physiology with the potential for translation to motile human cells, such as immune cells and spreading tumor cells.