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

所有的P2 X受体在静息膜电位下都有显著的Ca ~（2+）电流 触发细胞外的许多生理和病理生理作用 ATP这种Ca 2+电流的分子生理学知之甚少， 在功能和结构研究方面取得了重大进展。在本提案中，我们 专注于天然和重组P2 X7的Ca 2+电流的分子生理学 受体（P2 X7 Rs）。P2 X7 Rs在造血细胞中显著表达， 在炎症中起重要作用。它们也存在于骨骼、神经元和神经胶质中， 它们影响分化、稳态和退化。令人遗憾的是， 尽管经过40多年的深入研究，P2 X7 R文献中仍存在空白。在 在本提案的三个具体目标中概述的实验中，我们探索了两个未知数。 首先，我们试图确定比预期的Ca 2+的分子决定因素 大多数P2 X7 R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Ca 2+通量的特定结构域。这一目标意义重大，因为 从截短的P2 XR晶体导出的结构模型可能会呈现扭曲的视图 并且不能限定离子选择性过滤器。在具体目标2中， 我们调查了一个奇怪的发现，即自然发生的P2 X7 Rs的剪接变体， 不同的N-末端但相同的成孔螺旋导致显著不同的Ca 2 + 电流，这表明在门控期间移动的N-末端起着重要作用 在定位孔内Ca 2+选择性过滤器中。这些数据表明 与N末端相互作用的药物和信号复合物可以选择性地 调节生理上重要的Ca 2+通过通道的流量。第二，我们寻求 了解ATP电流随时间变化的生物物理基础 在长时间应用激动剂期间观察到逆转。传统上被认为反映了 毛孔逐渐扩张，最近的一份报告表明，逆转实际上发生在 离子重新分布穿过细胞表面膜。在第三部中，我们使用了新的野生动物- 型和突变型受体，以检验孔扩张和离子积累的假设， 并不是相互排斥的现象。这些实验很重要，因为 真正的孔扩张可以影响活细胞中的Ca 2+稳态。也就是说， 负责扩张的通道结构可能会破坏细胞内关键的Ca 2+结合位点， P2 X7 R孔，导致Ca 2+内流减少和细胞外ATP- 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.