Zelenina Marina, Zelenin Sergey, Bondar Alexander A, Brismar Hjalmar, Aperia Anita
Department of Woman and Child Health, Karolinska Institute, Astrid Lindgren Children's Hospital, S-171 76 Stockholm, Sweden.
Am J Physiol Renal Physiol. 2002 Aug;283(2):F309-18. doi: 10.1152/ajprenal.00260.2001.
Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P(f)) was measured in individual LLC-PK1 cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH2 terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased P(f) of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the P(f) in transfected cells. The effect was abolished by the PKC inhibitor Ro 31-8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser(180), a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser180 and that the effect is likely mediated by gating of the channel.
水通道蛋白4（AQP4）在集合管水的基底外侧转运中发挥重要作用。在此我们表明，这种水通道可被动态调节。在瞬时转染了AQP4的单个LLC-PK1细胞中测量水通透性（P(f)）。为了鉴定哪些细胞被转染，在AQP4的NH2末端用绿色荧光蛋白进行标记。转染细胞在基底外侧膜显示强荧光信号，而在细胞质和顶端膜显示低至可忽略不计的信号。用佛波醇12,13 -二丁酸酯（PDBu）激活蛋白激酶C（PKC）可显著降低表达AQP4的细胞的P(f)，但对相邻未转染细胞无影响。未检测到AQP4因PDBu发生重新分布。多巴胺也降低转染细胞中的P(f)。PKC抑制剂Ro 31 - 8220可消除该效应。PDBu和多巴胺对AQP4水通透性的降低作用可通过Ser(180)（PKC磷酸化的共有位点）的点突变而消除。我们得出结论，PKC和多巴胺通过Ser180磷酸化降低AQP4水通透性，且该效应可能由通道门控介导。
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