OPIOID AND G PROTEIN REGULATION OF CALCIUM CURRENTS

这项研究的总体目标是表征分子 阿片类药物抑制电压敏感性钙的作用机制 哺乳动物神经元上的(Ca~(2+))通道并确定它们是如何 受长期服用阿片类药物的影响。这项工作的前提是 最近的研究表明，Mu-阿片受体的激活 对kappa-阿片受体的刺激，抑制大鼠钙电流 背根节(DRG)感觉神经元依赖G蛋白 路径。一个主要目标是确定中国的压制是否 分别由Mu阿片受体和Kappa阿片受体产生的钙电流， 演示与以下通道类型相关的特性 被调节的和特定种类的G蛋白有关。至 要做到这一点，我们将记录急性分离的背根节中的钙电流 成年大鼠的神经元使用贴片的全细胞变异- 钳制技术和记录通过细胞内单个钙通道的电流- 贴膜贴片。Mu-和kappa-对钙离子通道的影响 阿片受体是偶联的将通过比较能力来鉴定 DAMGO和强啡肽或U69593分别抑制L、N和P- 全细胞电流的类型分量，由生物物理区分 标准及其对钙通道拮抗剂和激动剂的敏感性。 然后，我们将寻找相应的钙离子亚型的调制 沐浴时隔离在细胞贴片中的通道 用激动剂确定u-或kappa-阿片受体是否偶联到 钙离子通道通过可扩散的第二信使和/或膜分隔 信号通路。传统的第二信使在 阿片信号的转导也将通过比较 激动剂对早、晚期诱发的钙电流的影响 在用特定蛋白质进行细胞内透析的过程中 不同的G蛋白是否介导钙电流的抑制 Mu和kappa阿片受体。我们将尝试重建MU-AND PTX处理的神经元对Kappa阿片类药物的反应 重组G蛋白α亚基和比较一个小组的能力 特异性抗G/O/α和抗G/I/α抗体相互作用 然后将通过使用核内注射 选择性阻断特异性表达的反义寡核苷酸 A亚单位。在后来的研究中，适当的透析效果 将在吗啡耐受大鼠的神经元中测试G蛋白 完全和部分恢复钙电流抑制的能力 MU-阿片激动剂恢复到对照组的水平。吗啡，也许 涉及G蛋白的修饰，该G蛋白将u阿片受体偶联到 分子水平上的离子通道和转导，应该有助于 用于疼痛控制的治疗药物设计 阿片类药物的耐受性和滥用责任特征。

The overall goal of this research is to characterize the molecular mechanisms that mediate opioid inhibition of voltage-sensitive calcium (Ca2+) channels in mammalian neurons and to determine how they are affected by long-term opiate administration. The work is predicated on recent demonstrations that activation of mu-opioid receptors, in addition to kappa-opioid receptor stimulation, inhibits Ca2+ currents in rat dorsal root ganglion (DRG) sensory neurons via a G protein-dependent pathway. A major objective is to determine whether the suppression in Ca2+ current produced by mu- and kappa-opioid receptors, respectively, demonstrates specificity with respect to the types of channels that are modulated and the particular species of G proteins involved. To accomplish this, we will record Ca2+ currents in acutely isolated DRG neurons from adults rats using the whole-cell variation of the patch- clamp technique and record currents through single Ca2+ channels in cell- attached membrane patches. The Ca2+ channels to which mu-and kappa- opioid receptors are coupled will be identified by comparing the ability of DAMGO and dynorphin or U69593, respectively, to inhibit L-, N- and P- type components of whole-cell current, distinguished by biophysical criteria and their sensitivity to Ca2+ channels antagonists and agonists. We will then look for modulation of the corresponding subtypes of Ca2+ channels isolated in cell-attached patch during bath application of agonists to determine whether mu- or kappa-opioid receptors couple to Ca2+ channels via diffusible second messenger and/or a membrane delimited signaling pathway. The role of conventional second messengers in transduction of the opioid signal will also be assessed by comparing agonist-induced effects on Ca2+ currents evoked at early and late times during intracellular dialysis of the cell interior with specific protein whether distinct G proteins mediate the suppression in Ca2+ current by mu- and kappa-opioid receptors. We will attempt to reconstitute mu- and kappa-opioid responses in PTX-pretreated neurons using purified and recombinant G protein alpha subunits and compare the ability of a panel of specific anti-G/o/alpha and anti-G/i/alpha antibodies to interaction with Go will then be establish by using intranuclear injection of antisense oligonucleotide to selectivity block the expression of specific a subunits. In later studies, the effects of dialysis of the appropriate G protein will be tested in neurons from morphine-tolerate rats for their ability to reinstate the suppression in Ca+ current by full and partial mu-opioid agonists to the levels found in controls. The morphine, might involve modifications in G proteins that couple mu-opioid receptors to ion channels and transduction at the molecular level, should help in the design of therapeutic agents for pain management that possess little of the tolerance and abuse liability characteristic of opiate drugs.