Anesthetic Action:Channels Substrates & Mechanisms

吸入麻醉药是临床上广泛使用的有价值的麻醉剂。 然而，这些化合物引起临床重要作用（例如意识丧失和不动）的细胞和分子机制仍然不完全清楚。 越来越多的证据表明神经元膜离子通道是麻醉作用的直接靶点，历史上非常重视GABAA和甘氨酸受体。 我们的实验室最近已经证明，麻醉剂通过调节两种不同的离子通道来降低体运动神经元的兴奋性：激活背景或“泄漏”K+通道和抑制超极化激活的阳离子通道（Ih）。 这种通道调制发生在临床相关的浓度和运动神经元的抑制，结果可以解释，至少部分，麻醉剂的固定效果。 KCNK和HCN通道家族（神经元渗漏K+和Ih通道的底物）的相对较新的克隆为确定麻醉剂对这些通道的影响的分子机制提供了机会。 我们已发表的和初步的数据表明，运动神经元中的麻醉剂激活的K+电流涉及pH和神经递质敏感的ASK-1（KCNK 3）和ASK-3（KCNK 9）通道亚基，无论是在同源或异源配置;麻醉剂对这些泄漏K+通道的影响似乎是由神经递质作用调制。 同样，环核苷酸门控的HCN 1和HCN 2亚基在运动神经元中共表达，在那里它们也可以结合成同源或异源通道;我们的初步数据表明，挥发性麻醉剂不同地影响同源HCN亚基，cAMP调节麻醉剂的作用。 我们假设，挥发性麻醉剂对神经元漏钾电流和Ih的影响，以及神经递质对它们的调制，在克隆的ASK和HCN通道中完全重现，并且这些通道包括其一级结构内对麻醉作用至关重要的决定因素。 具体目标是：[1]阐明挥发性麻醉剂对“渗漏”K+（ASK）通道影响的分子机制; [2]阐明挥发性麻醉剂对超极化激活阳离子（HCN）通道影响的分子机制。 对于这些研究，我们记录克隆的ASK和HCN通道电流在哺乳动物异源表达系统和本地泄漏K+电流和Ih运动神经元。 我们表征麻醉剂的作用和它们的调制神经递质（或cAMP）克隆和天然通道，并使用定点诱变，以确定这些行动所必需的通道域。 这些实验将确定挥发性麻醉剂调节运动神经元的ASK和HCN通道的分子机制，并对其固定作用产生影响。 这些通道在中枢神经系统中的广泛表达表明，它们在其他脑区被麻醉剂调制可能有助于额外的麻醉作用。

Inhalation anesthetics are valuable agents in widespread clinical use. However, the cellular and molecular mechanisms by which these compounds elicit clinically important actions, such as loss of consciousness and immobility, are still incompletely understood. Accumulating evidence implicates neuronal membrane ion channels as direct targets for anesthetic effects, with much emphasis historically on GABAA and glycine receptors. Our laboratory has recently demonstrated that anesthetics decrease excitability in somatic motoneurons via modulation of two distinct ion channels: activation of background or 'leak' K+ channels and inhibition of hyperpolarization-activated cationic channels (Ih). This channel modulation occurs at clinically relevant concentrations and the motoneuronal inhibition that results could account, at least in part, for the immobilizing effects of anesthetics. The relatively recent cloning of KCNK and HCN channel families, the substrates for neuronal leak K+ and Ih, channels, provides an opportunity to determine molecular mechanisms underlying anesthetic effects on these channels. Our published and preliminary data indicate that the anesthetic- activated K+ current in motoneurons involves the pH- and neurotransmitter-sensitive TASK-1 (KCNK3) and TASK-3 (KCNK9) channel subunits, either in homo- or heteromeric configurations; anesthetic effects on these leak K+ channels appear to be modulated by neurotransmitter action. Likewise, the cyclic- nucleotide-gated HCN1 and HCN2 subunits are co-expressed in motoneurons, where they also may associate into homo- or heteromeric channels; our preliminary data indicate that volatile anesthetics affect homomeric HCN subunits differentially and that cAMP modulates effects of anesthetic. We hypothesize that the effects of volatile anesthetics on neuronal leak K+ currents and Ih, and their modulation by neurotransmitters, are fully recapitulated in cloned TASK and HCN channels, and that these channels include determinants critical for anesthetic effects within their primary structure. The Specific Aims are: [1] Elucidate molecular mechanisms underlying volatile anesthetic effects on 'leak' K+ (TASK) channels; [2] Elucidate molecular mechanisms underlying volatile anesthetic effects on hyperpolarization-activated cationic (HCN) channels. For these studies, we record cloned TASK and HCN channel currents in a mammalian heterologous expression system and native leak K+ currents and Ih in motoneurons. We characterize anesthetic effects and their modulation by neurotransmitters (or cAMP) on cloned and native channels, and use site-directed mutagenesis in order to identify channel domains that are necessary for these actions. These experiments will determine molecular mechanisms by which volatile anesthetics modulate TASK and HCN channels native to motoneurons, with implications for their immobilizing actions. Widespread expression of these channels in the CNS suggests that their modulation by anesthetics in other brain regions may contribute to additional anesthetic actions.