Mechanisms of Pannexin Channel Activation and permeation

项目4项目概要 Pannexin 1（Panx 1）是一种广泛表达的膜离子通道，当被激活时，导致跨膜 大分子的通量（即，核苷酸，其他代谢物），可以介导多种细胞间信号传导， （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是至关重要的 揭示途径特异性药理学干预新的潜在治疗策略的重要性， 可以选择性地调节特定信号代谢物在不同环境中的渗透。 在公认的Panx 1激活机制中，G α q蛋白偶联受体介导的 （G α qPCR）是广泛的，但介导这一点的基本细胞，分子和生物物理机制， 尚未阐明通道激活的普遍形式。我们的初步数据表明， 激酶，SIK1，一种丝氨酸-苏氨酸激酶，与Panx1物理结合，是必需的， 足以激活通道。在目标1中，通过额外的初步观察，我们测试了 假设G α qPCR通过涉及LKB 1和RhoA-mDia-HDAC 6的非经典途径进行信号传导， 汇聚激活SIK1介导磷酸化和激活Panx1。为此，我们使用基因和 药理学工具，在异源和天然系统中，以确定相关的信号传导途径， 通过突变、质谱和体外激酶方法鉴定关键通道磷酸化位点。在 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 类型和串联Panx1结构，检查通道是否以新颖的逐步方式激活 我们最近发现的C端裂解激活通道。 Panx1通道以其与核苷酸释放和染料摄取的关联而闻名。尽管如此， 还没有确定这些大分子是否真的通过通道本身渗透，甚至 Panx 1的离子选择性尚未确定。此外，由不同机制激活的通道 显示出独特的单通道特性，这表明它们也可以产生独特的渗透特性， 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， 纯化的Panx 1，以测试活化的Panx 1提供支持释放的渗透途径的假设 不同的细胞成分，以及不同的分子流量是由不同的通道模式的影响， activation.通过直接测量特定信号传导代谢物通过这些纯化的Panx 1的渗透， 通道，我们将确定代谢物的范围，可以通过通道时，激活半胱天冬酶， 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调节的分子机制， 确定渗透性能和信号代谢物支持的特定激活机制。

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.