Mechanisms of Pannexin Channel Activation and permeation

项目4项目总结 PAnnexin 1(Panx1)是一种广泛表达的膜离子通道，当被激活时，会导致跨膜 大分子(即核苷酸、其他代谢物)的通量，可以在多个 (病态)生理环境(例如，见项目1-3)。因此，了解不同的细胞和分子 通道激活的机制和大分子渗透的决定因素至关重要。 揭示通路特异性药物干预的新的潜在治疗策略的重要性 可以在不同的环境中选择性地调节特定信号代谢物的渗透。 在已建立的由G-αQ蛋白偶联受体介导的Panx1激活机制中， (GαqPCRs)广泛存在，但介导这一过程的基本细胞、分子和生物物理机制 通道激活的普遍形式尚未阐明。我们的初步数据表明盐诱导的 SIK1，一种丝氨酸-苏氨酸激酶，与Panx1物理上结合，是必需的，也是 足以激活通道。在目标1中，在其他初步观察的支持下，我们测试了 假设GαqPCRs信号通过涉及LKB1和RhoA-mdia-HDAC6的非规范途径，这是 聚合以激活SIK1，介导Panx1的磷酸化和激活。为此，我们使用基因和 药理工具，在异源和本地系统中，以确定相关的信号通路和 通过突变、质谱学和体外激酶法鉴定关键通道亚磷酸酶。在……里面 此外，我们使用单通道记录来表征部分和完全受体激活的野生型的特性 类型和串联的Panx1构造，检查通道是否以新颖的逐步方式激活 这是我们最近发现的C末端裂解激活的通道。 Panx1通道以其与核苷酸释放和染料摄取有关而闻名。尽管如此，它 尚未确定这些大分子是否真的通过通道本身渗透，甚至是 Panx1的离子选择性尚未确定。此外，不同机制激活的通道 显示不同的单通道属性，这表明它们也可能产生不同的渗透属性， 支持特定信号分子的释放。在目标2中，我们实现了一个蛋白脂质体系统 纯化Panx1以测试激活Panx1提供支持释放的渗透途径的假设 不同的细胞成分，不同分子的流量受到不同的通道模式的影响 激活。通过直接测量特定信号代谢物通过这些纯化的Panx1的渗透性 通道，我们将确定当被caspase激活时可以转运通道的代谢物的范围- 介导的C末端切割或SIK1介导的磷酸化。 这项工作定义了生理相关形式的Panx1调控的分子机制，以及 确定特定激活机制支持的渗透特性和信号代谢物。

PROJECT 4 PROJECT SUMMARY Pannexin 1 (Panx1) is a widely-expressed membrane ion channel that, when activated, leads to transmembrane flux of large molecules (i.e., nucleotides, other metabolites) that can mediate intercellular signaling in multiple (patho)physiological contexts (e.g., see Projects 1-3). Thus, understanding the different cellular and molecular mechanisms for channel activation, and the determinants for large molecule permeation, are of paramount importance to reveal novel potential therapeutic strategies for pathway-specific pharmacological intervention that could selectively modulate permeation of specific signaling metabolites in different contexts. Among well-established Panx1 activation mechanisms, that mediated by Gαq protein-coupled receptors (GαqPCRs) is widespread, but the essential cellular, molecular and biophysical mechanisms that mediate this prevalent form of channel activation have not been elucidated. Our preliminary data implicate the salt-inducible kinase, SIK1, a serine-threonine kinase that physically associates with Panx1, and is both necessary and sufficient for channel activation. In Aim 1, supported by additional preliminary observations, we test the hypothesis that GαqPCRs signal via non-canonical pathways involving LKB1 and RhoA-mDia-HDAC6, which converge to activate SIK1 to mediate phosphorylation and activation of Panx1. For this, we use genetic and pharmacological tools, in heterologous and native systems, to determine the relevant signaling pathways and identify critical channel phosphosites by mutational, mass spectrometric and in vitro kinase approaches. In addition, we use single channel recordings to characterize properties of partially and fully receptor-activated wild type and concatenated Panx1 constructs, examining whether channels activate in the novel stepwise fashion that we recently discovered for C-terminally cleavage-activated channels. Panx1 channels are renowned for their association with nucleotide release and dye uptake. Nonetheless, it has not been established whether these large molecules actually permeate via the channel itself, and even the ionic selectivity of Panx1 has not been established. In addition, channels activated by different mechanisms display distinct single channel properties, suggesting that they may also yield distinct permeation properties that support release of specific signaling molecules. In Aim 2, we implement a proteoliposome system incorporating purified Panx1 to test the hypothesis that activated Panx1 provides a permeation pathway that supports release of various cellular constituents, and that flux of different molecules is influenced by distinct modes of channel activation. By directly measuring permeation of specific signaling metabolites through these purified Panx1 channels, we will identify the range of metabolites that can transit the channel when activated by either caspase- mediated C-terminal cleavage or SIK1-mediated phosphorylation. This work defines molecular mechanisms underlying physiologically relevant forms of Panx1 regulation, and identifies permeation properties and signaling metabolites supported by specific activation mechanisms.