Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels
Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels
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DOI：
10.1152/ajpcell.00042.2003
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发表时间：
2003-08-01
影响因子：
5.5
通讯作者：
Stojilkovic, SS
中科院分区：
文献类型：
--
作者：
He, ML;Zemkova, H;Stojilkovic, SS
细胞外核苷酸激活的嘌呤能受体（P2 XRs）是阳离子渗透性通道家族，其传导包括Ca 2+的小阳离子，导致细胞去极化和随后刺激可兴奋细胞中的电压门控Ca 2+内流。在这里，我们研究了细胞内Ca 2+信号的时空特征及其依赖于电流信号在可兴奋的小鼠永生化的促性腺激素释放激素分泌细胞（GT 1）和非兴奋的人胚肾细胞（HEK-293）细胞表达野生型和嵌合P2 XRs。在这两种细胞类型中，P2 XR在持续ATP刺激期间产生去极化电流，其脱敏顺序（从快速脱敏到非脱敏）：P2 X（3）R > P2 X（2b）+X4 R> P2 X（2b）R > P2 X（2a）+X4 R> P2 X（4）R > P2 X（2a）R > P2 X（7）R。HEK-293细胞不适合研究P2 XR介导的Ca 2+内流，因为内源性表达的Ca 2+动员嘌呤能P2 Y受体的共激活。然而，当在GT 1细胞中表达时，所有野生型和嵌合P2 XR都对与全局Ca 2+信号结合的激动剂作出反应，其以与电流信号相同的顺序脱敏，但以明显较慢的方式脱敏。钙离子信号的全球分布是目前独立的电流脱敏率。电压门控性Ca 2+内流和细胞外钠清除不影响Ca 2+信号的时间特征。Ca 2+信号很好地反映了ATP的受体特异性EC 50值以及P2 XRs对胞外Zn 2+和pH的敏感性。这些结果表明，当宿主细胞不表达其他嘌呤能受体成员时，细胞内Ca 2+测量可用于表征P2 XR的药理学特性和信使功能以及通道活性动力学。
Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X(3)R > P2X(2b) + X4R > P2X(2b)R > P2X(2a) + X4R > P2X(4)R > P2X(2a)R > P2X(7)R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.