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

我们已经鉴定了一种新的水通道蛋白，AqpB，来自盘状盘齿龙。AqpB具有水特异性，o -糖基化，并表现出一种新的门控机制。它局限于液泡结构、质膜和膜突起。到目前为止，它是盘形钢索菌中唯一具有功能的水通道蛋白，在所有发育阶段都有表达。门控的关键结构域包括d环中的7个氨基酸位置。到目前为止，基因敲除的尝试已经产生了非特异性基因组整合。除了AqpB，我们还在cDNA和蛋白水平上鉴定出了变形虫体内的第二个水通道蛋白AqpC。下面我们将进一步阐明AqpB的调控机制，主要分析AqpB的生理功能，初步分析AqpC的生理功能。我们将产生新的AqpB突变，特别是在保守的酪氨酸-218附近的序列中进行生物物理分析。我们将构建具有更长的连接臂的敲除质粒，并选择AqpB编码区内富含gc的序列，以提高基因组整合的特异性。如果基因敲除是致命的，我们将采用琥珀色抑制敲除方法，将蛋白质水平降低高达90%。通过使用去调控aqpb变体的显性过表达，我们将测试门控在细胞生理学方面的作用。我们将分析敲除/敲低/过表达培养的表型，包括形态学、细胞分裂、发育、运动性、体积调节和巨噬细胞增多症。我们期望获得关于水通道蛋白在细胞生理学中的作用的基本知识，并有可能将其转化为活动的人类细胞，如免疫细胞和扩散的肿瘤细胞。

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.