Effects of cyclic nucleoticles on the function of prestin
Effects of cyclic nucleoticles on the function of prestin
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DOI：
10.1113/jphysiol.2004.078857
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发表时间：
2005-03-01
影响因子：
5.5
通讯作者：
Dallos, P
中科院分区：
文献类型：
--
作者：
Deák, L;Zheng, J;Dallos, P
哺乳动物Corti器官中的外毛细胞（OHC）显示电运动性，其被认为提供耳蜗反应的局部主动机械放大。普雷斯廷是负责OHC电活动的关键分子。几种化合物，包括cGMP，已被证明会影响OHC电活动。在普雷斯廷上存在两个潜在的cAMP/cGMP依赖性蛋白激酶磷酸化位点。这些位点是否参与cGMP依赖性反应尚不清楚。本研究将普雷斯廷cDNA瞬时转染TSA 201细胞。选择表达普雷斯廷的细胞来测量非线性电容（NLC），这是外毛细胞运动性的标志。我们应用cGMP和cAMP类似物和蛋白激酶G（PKG）拮抗剂的细胞。此外，在推定的磷酸化位点的普雷斯廷产生了9个突变。中性氨基酸丙氨酸取代丝氨酸/苏氨酸的磷酸化位点，改变保守的磷酸化基序，以模拟去磷酸化状态的普雷斯廷，而取代带负电荷的天冬氨酸模拟磷酸化状态。通过测量NLC和用共聚焦显微镜检查这种修饰的prestin表达细胞的性质。我们的数据表明，cGMP是显着更有影响力比cAMP在修改非线性，电压依赖性的电荷位移prestin转染细胞。单突变和双突变的电特性进一步表明两个PKG靶位点之间可能存在相互作用。这些位点之一可能影响普雷斯廷的膜靶向过程。最后，提出了一种新的普雷斯廷拓扑图。
Outer hair cells (OHCs) in the mammalian organ of Corti display electromotility, which is thought to provide the local active mechanical amplification of the cochlear response. Prestin is the key molecule responsible for OHC electromotility. Several compounds, including cGMP, have been shown to influence OHC electromotility. There are two potential cAMP/cGMP-dependent protein kinase phosphorylation sites on prestin. Whether these sites are involved in cGMP-dependent reactions is as yet unknown. In this study, prestin cDNA was transiently transfected into TSA 201 cells. Cells that expressed prestin were selected to measure non-linear capacitance (NLC), a signature of outer hair cell motility. We applied cGMP and cAMP analogues and a protein kinase G (PKG) antagonist to the cells. Furthermore, nine mutations at putative phosphorylation sites of prestin were produced. The neutral amino acid alanine replaced serine/threonine at phosphorylation sites to change the conserved phosphorylation motif in order to mimic the dephosphorylated state of prestin, whereas replacement with the negatively charged aspartic acid mimicked the phosphorylated state. The properties of such modified prestin-expressing cells were examined, through measurement of NLC and with confocal microscopy. Our data demonstrate that cGMP is significantly more influential than cAMP in modifying the non-linear, voltage-dependent charge displacement in prestin-transfected cells. The electrical properties of the single and double mutations further indicate a possible interaction between the two PKG target sites. One of these sites may influence the membrane targeting process of prestin. Finally, a new topology map of prestin is proposed.