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

ATP 门控 P2X7 电流钙成分的选择性调节

• 批准号: 9196585
• 负责人: TERRANCE M EGAN
• 金额: $ 30.3万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196585
• 关键词: Address; Agonist; Autoimmune Diseases; Binding; Binding Sites; Biology; Biophysics; Calcium; Cardiac; Cations; Cell physiology; Cell surface; Cells; Communities; Complex; Cytoplasm; Cytoplasmic Tail; Data; Dementia; Endocrine Glands; Family; Feedback; Goals; Hematopoietic; Histamine; Homeostasis; Hormones; Immune; Immune response; Inflammation; Ion Channel; Ions; Lateral; Left; Life; Literature; Measures; Membrane; Membrane Potentials; Methods; Molecular; Muscle; Neuroglia; Neurons; Outcome; Outer Leaflet of the Lipid Bilayer; P2X-receptor; Pathway interactions; Permeability; Pharmaceutical Preparations; Photometry; Physiological; Physiology; Play; Positioning Attribute; Property; Proteins; RNA Splicing; Receptor Activation; Recombinants; Regulation; Reporting; Research; Rest; Role; Signal Transduction; Structural Models; Structure; Testing; Time; Vacuole; Variant; Width; base; bone; cytokine; desensitization; design; extracellular; fascinate; innovation; interest; mutant; new therapeutic target; novel; painful neuropathy; patch clamp; receptor; research study; skeletal

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.

所有 P2X 受体在静息膜电位下均能转导显着的 Ca2+ 电流 触发细胞外的许多生理和病理生理作用 ATP。尽管人们对这种 Ca2+ 电流的分子生理学知之甚少 功能和结构研究的最新重大进展。在这个提案中，我们 专注于天然和重组 P2X7 的 Ca2+ 电流的分子生理学 受体（P2X7R）。 P2X7R 在造血细胞中显着表达并发挥作用 在炎症中发挥重要作用。它们还存在于骨骼、神经元和神经胶质中 它们影响分化、稳态和退化。遗憾的是，有意义 尽管经过 40 多年的深入研究，P2X7R 文献中仍然存在空白。在 在该提案的三个具体目标中概述的实验中，我们探讨了两个未知数。 首先，我们寻求确定高于预期的 Ca2+ 的分子决定因素 大多数 P2X7R 的通量。在具体目标 1 中，我们使用膜片钳光度测定法来识别 负责 Ca2+ 通量的特定域。这一目标意义重大，因为 从截短的 P2XR 晶体衍生的结构模型可能会呈现扭曲的视图 渗透路径并且未能定义离子选择性过滤器。在具体目标 2 中， 我们研究了一个奇怪的发现，即 P2X7R 的自然发生的剪接变体 不同的 N 末端但相同的成孔螺旋转导显着不同的 Ca2+ 电流，表明在门控过程中移动的 N 末端发挥着重要作用 定位孔内 Ca2+ 选择性过滤器。这些数据留下了可能性 与 N 末端相互作用的药物和信号复合物可能选择性地 调节生理上重要的 Ca2+ 通过通道的通量。其次，我们寻求 了解 ATP 电流随时间变化的生物物理基础 长期应用激动剂时出现逆转。传统上认为反映 随着毛孔逐渐扩张，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。在特定目标 3 中，我们使用新颖的野生 类型和突变受体来检验孔扩张和离子积累的假设 不是相互排斥的现象。这些实验很重要，因为 真正的毛孔扩张可能会影响活细胞中的 Ca2+ 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关键的 Ca2+ 结合位点 P2X7R 孔，导致 Ca2+ 内流减少和细胞外 ATP- 变化 依赖细胞生理学。