Intracellular Signaling In Endocrine Cells

我们从脑垂体中克隆了5个atp门控的P2X受体通道（P2XRs）： P2X2R、P2X3R、P2X4R、P2X6R和P2X7R。我们正在进行的工作集中在它们的结构功能表征上。当在下丘脑GT1细胞中表达时，P2X7R的激活诱导了一个可渗透到小阳离子的完整离子通道的快速打开。随后是对荧光染料的渗透性逐渐增加。P2X7R的这种双功能渗透特性可能反映了通道整体孔的扩张或激活通道（如泛联蛋白）整合了另一种渗透途径。我们提供了几条证据表明P2X7R孔隙扩张：1。在长时间的激动剂应用过程中，一个在200毫秒内达到峰值的快速电流伴随着一个需要几十秒才能达到峰值的较慢电流。2. 在不同的离子条件下观察到电流的二次上升，并且在时间上与电导率的发展相一致。3. 在泛联蛋白通道被阻断的细胞和不内源性表达这些通道的细胞中也观察到双相反应。4. 在对有机阳离子渗透率低或无渗透率的n端T15A、T15S和T15V突变体中，双相电流得以保留，反映了对无机阳离子的渗透率增强。相比之下，T15E、T15K和T15W突变体以及缺失p2x7r特异性18个氨基酸c端片段的D18突变体能够瞬时渗透有机阳离子并产生高振幅单相电流。综上所述，这些结果表明P2X7R通道在生理离子条件下扩张，导致双相电流的产生，而这一过程是由通道孔细胞内侧附近的残基控制的。我们还研究了P2XRs跨膜结构域-1上部芳香残基的功能相关性。用Ala取代保守的Tyr残基具有受体特异性效应：P2X1R无功能，P2X2R、P2X4R和P2X3R对ATP和-meATP的敏感性增强，并伴随着激动剂洗掉后电流衰减时间的延长，而P2X7R对激动剂的敏感性不受影响，但电流衰减延迟。用其他氨基酸替换P2X4R-Tyr42显示了在该位置上的芳香残基的相关性。邻近的Phe和同侧的Tyr/Trp残基的突变也会影响P2X2R、P2X3R和P2X4R的功能，而对侧的Phe残基则不会。同侧Tyr42和Trp46 P2X4R残基的双突变恢复了受体功能，而相应的P2X2R双突变则没有功能。相比之下，P2X4R-Y42A突变体中对侧Phe48残基的突变没有影响。这些结果表明，TM1上部的芳香残基在p2xr的三维结构中起着重要作用，它们不仅是离子电导率所必需的，而且也是激动剂结合和/或通道门控的特异性所必需的。我们与约翰霍普金斯大学的神经生物系合作，研究了神经元细胞中β -淀粉样蛋白和P2X4R之间的相互作用。除此之外，这些实验揭示了β -淀粉样蛋白片段1-42诱导了caspase-3介导的受体裂解，从而减缓了通道关闭时间并阻止了激动剂诱导的受体内化。沉默内源性P2X4R的表达可减轻β -淀粉样蛋白片段1-42诱导的神经元死亡，而在通常不表达P2XRs的细胞系中表达P2X4R可增强该片段的毒性作用。这些发现表明，β -淀粉样蛋白诱导的突触功能障碍和神经元死亡可能涉及P2XR转运和功能的改变。我们与智利圣地亚哥天主教大学合作，重点研究了几种主要作用于受体外结构域的化合物对P2X2R的变构调节。像铜一样，汞，一种在细胞中引起氧化应激的金属，也会刺激P2X2R的活性并抑制P2X4R的活性。然而，汞调制与对铜调制至关重要的细胞外残基无关。为了确定汞作用位点，我们使用全尺寸P2X2亚基（称为P2X2a）和c端缺乏69个残基片段的剪接变体（称为P2X2b）作为嵌合体的细胞内和跨膜片段的供体，并使用P2X4亚基作为嵌合体的外结构域片段的供体，生成了两个嵌合体。汞对atp诱导电流的增强作用在表达P2X4/2a嵌合体的爪蟾卵母细胞中保留，而在表达P2X4/2b嵌合体的卵母细胞中不存在。位点诱变实验表明，Cys-430残基介导汞对P2X2aR活性的影响。由于汞可以作为氧化应激诱导剂，我们还测试了过氧化氢和线粒体应激诱导剂粘噻唑和鱼tenone是否模拟汞的作用。这些实验表明，这些化合物增强了atp引起的P2X2aR和P2X4/2aR电流，但对P2X2bR、P2X2a-C430A和P2X2a-C430S突变电流没有作用，而抗氧化剂二硫代苏硝基和n -乙酰半胱氨酸则阻止了过氧化氢的增强。Cys-430残基与甲基甲烷-硫代磺酸盐的烷基化反应也消除了汞和过氧化氢的增强作用。总之，这些结果与Cys-430残基是细胞内P2X2aR氧化还原传感器的假设是一致的。通过与NICHD分子信号转导部门的合作，我们的团队还对STIM1/ orai1介导的钙进入的表征做出了贡献。我们在这个合作项目中的工作重点是研究I-crac电流对STIM1/ orai1表达细胞中磷酸肌苷的依赖性。我们发现wortmannin对III型PI4激酶的抑制作用强烈影响I-crac电流的振幅。我们还发现，血管紧张素II受体激活磷脂酶C信号通路导致I-crac电流的快速但不完全抑制。这些结果表明，PtdIns4P而不是PtdIns(4,5)P2可能是Orai通道活性的决定因素。

We have cloned five ATP-gated P2X receptor channels (P2XRs) from the pituitary gland: P2X2R, P2X3R, P2X4R, P2X6R, and P2X7R. Our ongoing work is focused on their structural-functional characterization. When expressed in hypothalamic GT1 cells, activation of the P2X7R induced the rapid opening of an integral ion channel that was permeable to small cations. This was followed by a gradual increase in permeability to fluorescent dyes. Such bi-functional permeation properties of P2X7R could reflect a dilation of integral pore of the channels or integration of another permeation pathway by activated channels, such as pannexins. We provided several lines of evidence indicating that the P2X7R pore dilates: 1. During the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. 2. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. 3. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. 4. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the D18 mutant with deleted P2X7R-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. Together, these results indicate that the P2X7R channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore. We also studied the functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of P2XRs. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was nonfunctional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and αβ-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating. In collaboration with the Department of Neurobiology from Johns Hopkins University, we have also worked on interactions between beta-amyloid and P2X4R in neuronal cells. Among others, these experiments revealed that the beta-amyloid fragment 1-42 induced a caspase-3-mediated cleavage of the receptor that slowed channel closure times and prevented agonist-induced internalization of the receptor. Silencing the expression of endogenous P2X4R attenuated the beta-amyloid fragment 1-42-induced neuronal death, while expression of P2X4R in a cell line that does not normally express P2XRs enhanced the toxic effects of this fragment. These findings suggested that beta-amyloid-induced synaptic dysfunction and neuronal death may involve alternations in P2XR trafficking and functions. Our collaborative work with Catholic University in Santiago, Chile, was focused on allosteric modulation of P2X2R by several compounds, mainly acting at receptors ectodomain. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69-residue segment in the C-terminal, termed P2X2b, as donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera, but was absent in oocytes expressing P2X4/2b chimera. Site directed mutagenesis experiments revealed that the Cys-430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide and mitochondrial stress inducers myxothiazol, and rotenone mimicked mercury effects. These experiments revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents, but not P2X2bR and P2X2a-C430A and P2X2a-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the hydrogen peroxide-potentiation. Alkylation of Cys-430 residue with methylmethane-thiosulfonate also abolished the mercury and hydrogen peroxide potentiation. Altogether, these results are consistent with the hypothesis that the Cys-430 residue is an intracellular P2X2aR redox sensor. In collaboration with the Section on Molecular Signal Transduction of NICHD, our group has also contributed to the characterization of STIM1/Orai1-mediated calcium entry. The focus in our work in this collaborative project was on the dependence of I-crac current on phosphoinositides in STIM1/Orai1-expressing cells. We showed that the inhibition of type III PI4 kinase by wortmannin strongly affected the amplitude of the I-crac current. We also show that activation of phospholipase C signaling pathway by angiotensin II receptors caused rapid but incomplete inhibition of I-crac current. These results indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai channel activity.