Mockett B G, Housley G D, Thorne P R
Department of Physiology, School of Medicine, University of Auckland, New Zealand.
J Neurosci. 1994 Nov;14(11 Pt 2):6992-7007. doi: 10.1523/JNEUROSCI.14-11-06992.1994.
Fluorescence imaging of extracellular adenosine-5'-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea pig organ of Corti was achieved using the fluorescent analog of ATP, 2'-(or-3')-O-(trinitrophenyl)adenosine-5'- triphosphate (TNP-ATP; 30-75 microM). This analog, which fluoresces on binding to these sites, was pressure applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution of extracellular binding sites, including the stereocilia, cuticular plate, and the basolateral margins of the cells, but particularly in infracuticular and infranuclear regions. In support of extracellular binding, simultaneous electrophysiological recordings demonstrated that rapid washout of TNP-ATP-induced fluorescence was dependent upon cell integrity. Suramin, a nonselective P2 purinoceptor antagonist, coapplied with TNP-ATP, reduced the fluorescence observed on the stereocilia and apical surface of the cuticular plate only. This implies that binding sites on the apical surface of hair cells are P2 receptors, consistent with previous electrophysiological evidence for localization of P2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P2 purinoceptors was confirmed by its antagonism of the inward current elicited by ATP (10 microM) in voltage-clamped hair cells. Fluorescence from the basolateral margin was significantly quenched when TNP-ATP was applied in divalent cation-free solution. Because divalent cations are required for ATPase activity, this finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The divalent cation-free condition had no significant effect on the ATP-gated P2 purinoceptor conductance. We propose that there are two classes of ATP binding sites on cochlear hair cells: apically located P2 purinoceptors gating nonselective cation channels and basolaterally located ecto-ATPases that may be involved in purine turnover.
利用三磷酸腺苷（ATP）的荧光类似物2'-（或3'）-O-（三硝基苯基）三磷酸腺苷（TNP-ATP；30 - 75微摩尔），对从豚鼠柯蒂氏器分离出的内、外毛细胞上的细胞外ATP结合位点进行了荧光成像。这种在与这些位点结合时会发出荧光的类似物，通过微量移液器施加压力，同时用荧光显微镜观察毛细胞。荧光成像显示细胞外结合位点分布广泛，包括静纤毛、角质板以及细胞的基底外侧边缘，但在角质层下和核下区域尤为明显。为支持细胞外结合，同时进行的电生理记录表明，TNP-ATP诱导的荧光快速洗脱取决于细胞完整性。苏拉明是一种非选择性P2嘌呤受体拮抗剂，与TNP-ATP共同施加时，仅降低了在静纤毛和角质板顶端表面观察到的荧光。这意味着毛细胞顶端表面的结合位点是P2受体，这与先前关于P2受体定位于耳蜗毛细胞顶端表面的电生理证据一致（豪斯利等人，1992年）。TNP-ATP与P2嘌呤受体的结合通过其对电压钳制的毛细胞中ATP（10微摩尔）引发的内向电流的拮抗作用得到证实。当在无二价阳离子的溶液中施加TNP-ATP时，基底外侧边缘的荧光明显淬灭。由于二价阳离子是ATP酶活性所必需的，这一发现为毛细胞基底外侧膜上存在胞外ATP酶提供了证据。无二价阳离子条件对ATP门控的P2嘌呤受体电导没有显著影响。我们提出，耳蜗毛细胞上存在两类ATP结合位点：顶端定位的P2嘌呤受体，其开启非选择性阳离子通道；基底外侧定位的胞外ATP酶，可能参与嘌呤周转。
