Anesthetic Action:Channels Substrates & Mechanisms

吸入麻醉剂是临床上广泛使用的有价值的药物。然而，这些化合物引发临床重要行为的细胞和分子机制，如意识丧失和不活动，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，历史上非常强调GABAA和甘氨酸受体。我们的实验室最近证明，麻醉剂通过调节两种不同的离子通道来降低躯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）。这种通道调节发生在临床相关浓度和运动神经元抑制的结果可以解释，至少部分，麻醉药的固定作用。最近克隆的KCNK和HCN通道家族（神经元泄漏K+和Ih通道的底物）为确定麻醉作用于这些通道的分子机制提供了机会。我们发表的和初步的数据表明，麻醉激活的K+电流在运动神经元中涉及pH和神经递质敏感的TASK-1 （KCNK3）和TASK-3 （KCNK9）通道亚基，无论是同质构型还是异质构型；麻醉对这些泄漏K+通道的影响似乎是由神经递质作用调节的。同样，环核苷酸门控的HCN1和HCN2亚基在运动神经元中共同表达，它们也可能结合到同质或异质通道中；我们的初步数据表明，挥发性麻醉剂对同源HCN亚基的影响是不同的，cAMP调节了麻醉剂的作用。我们假设，挥发性麻醉药对神经元泄漏K+电流和Ih的影响，以及神经递质对它们的调节，在克隆的TASK和HCN通道中得到了充分的概括，并且这些通道在其初级结构中包括对麻醉作用至关重要的决定因素。具体目标是：阐明挥发性麻醉对“泄漏”K+ (TASK)通道影响的分子机制；阐明挥发性麻醉药对超极化活化阳离子（HCN）通道影响的分子机制。在这些研究中，我们记录了哺乳动物异种表达系统中克隆的TASK和HCN通道电流，以及运动神经元中天然泄漏的K+电流和Ih。我们描述了麻醉效应及其由神经递质（或cAMP）对克隆和天然通道的调节，并使用位点定向诱变来确定这些作用所必需的通道域。这些实验将确定挥发性麻醉剂调节运动神经元固有的TASK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，麻醉剂在其他脑区对它们的调节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.