Selective regulation of the calcium component of the ATP-gated P2X7 current

所有的P2X受体都在静息膜电位上传递显著的钙电流 触发了许多细胞外的生理和病理生理活动 三磷酸腺苷。尽管人们对这种钙电流的分子生理学知之甚少 最近在功能和结构研究方面取得了重大进展。在这项提案中，我们 关注天然和重组P2X7钙电流的分子生理学 受体(P2X7Rs)。P2X7Rs在造血细胞中显著表达并发挥作用 在炎症中起着至关重要的作用。它们也存在于骨骼、神经元和神经胶质细胞中 它们影响分化、动态平衡和退化。遗憾的是，这是有意义的 尽管进行了40年的密集研究，但P2X7R文献中仍然存在空白。在 在这项提议的三个具体目标中概述的实验，我们探索了两个未知数。 首先，我们试图确定超出预期的钙离子的分子决定因素。 大多数P2X7Rs的通量。在具体目标1中，我们使用膜片钳光度法来识别 负责钙通量的特定结构域。这一目标意义重大，因为 从截断的P2XR晶体导出的结构模型可能呈现扭曲的观点 并且没有定义离子选择性过滤器。在具体目标2中， 我们调查了一个奇怪的发现，自然发生的P2X7Rs剪接变体与 不同的N末端但相同的致孔螺旋转导显著不同的钙 电流，这表明在选通过程中移动的N末端起着重要的作用 在定位孔内钙选择性过滤器方面。这些数据保留了一种可能性 与N末端相互作用的药物和信号复合体可能选择性地 通过该通道调节生理上重要的钙离子流量。第二，我们寻求 了解ATP电流随时间变化的生物物理基础 在长期使用激动剂时出现逆转。传统上认为反映了一种 毛孔逐渐扩张，最近的一份报告表明，这种逆转实际上发生在 离子在细胞表面膜上重新分布。在具体目标3中，我们使用了新奇的狂野- 类型和突变的受体来检验孔扩张和离子积累的假说 并不是相互排斥的现象。这些实验之所以重要，是因为 真正的毛孔扩张可能会影响活细胞中的钙稳态。也就是说，修改后的 负责扩张的通道结构可能会破坏细胞内的一个关键的钙结合位点 P2X7R孔道，导致细胞内钙内流减少，细胞外ATP含量改变。 依赖细胞生理学。

All P2X receptors transduce significant Ca2+ currents at the resting membrane potential that trigger many of the physiological and pathophysiological actions of extracellular ATP. The molecular physiology of this Ca2+ current is poorly understood despite significant recent advances in functional and structural studies. In this proposal, we focus on the molecular physiology of the Ca2+ current of native and recombinant P2X7 receptors (P2X7Rs). P2X7Rs are prominently expressed in hematopoietic cells and play an essential role in inflammation. They are also found in bone, neurons, and glia where they influence differentiation, homeostasis, and degeneration. Regrettably, meaningful gaps remain in the P2X7R literature despite 40+ years of intensive study. In the experiments outlined in the three specific aims of this proposal, we probe two unknowns. First, we seek to identify the molecular determinates of the greater-than-expected Ca2+ flux of most P2X7Rs. In Specific Aim 1, we use patch-clamp photometry to identify specific domains responsible for the Ca2+ flux. This aim is significant because the structural models derived from crystals of truncated P2XRs may present a distorted view of the permeation pathway and fail to define the ion selectivity filter. In Specific Aim 2, we investigate the curious finding that naturally occurring splice variants of P2X7Rs with distinct N-termini but identical pore-forming helices transduce dramatically different Ca2+ currents, suggesting that the N-termini, which move during gating, play a significant role in positioning an intra-pore Ca2+ selectivity filter. These data leave open the possibility that drugs and signaling complexes that interact with the N-terminus might selectively modulate the physiologically important Ca2+ flux through the channel. Second, we seek to understand the biophysical basis of the time-dependent changes in ATP-current reversal seen during long applications of agonist. Traditionally thought to reflect a gradual dilation of the pore, a recent report suggests that the reversal actually occurs as ions redistribute across the cell surface membrane. In Specific Aim 3, we use novel wild- type and mutant receptors to test the hypothesis that pore dilation and ion accumulation are not mutually exclusive phenomena. These experiments are important because genuine pore dilation could impact Ca2+ homeostasis in living cells. That is, the modified channel structure responsible for dilation could disrupt a key Ca2+ binding site within the P2X7R pore, leading to a reduction in Ca2+ influx and a change in extracellular ATP- dependent cell physiology.