ULTRASTRUCTURE AND FUNCTION OF NERVE AND MUSCLE

描述(申请人提供)：离子通道是神经元兴奋性变化的主要决定因素。我们的长期目标是阐明脑神经元缓慢兴奋和缓慢抑制发生的机制。这些事件在几十秒到几分钟的时间尺度上发生，主要由G蛋白介导。我们的近期研究将集中于阐明兴奋性多肽神经递质P物质(SP)对G蛋白偶联内向整流Kv(GIRK)通道的信号转导机制。将使用新生大鼠和小鼠蓝斑(LC)中天然培养的去甲肾上腺素能神经元，以及在HEK293细胞中表达的克隆GIRK。将采用电生理和分子生物学技术进行研究。LC含有为大脑的大片区域提供去甲肾上腺素的神经元，在唤醒和警觉方面起着至关重要的作用。此外，在阿尔茨海默病中，LC神经元经常退化。LC神经元受到相反信号的双重调节：抑制性递质(如生长抑素)激活GIRK通道，而兴奋性递质(如SP)抑制由生长抑素激活的GIRK活性。然而，抑制GIRK的机制仍存在争议，尚未确定。本研究的目的是阐明SP诱导GIRK通道抑制的信号转导机制。SP诱导的GIRK通道抑制有三条可能的信号转导途径：磷脂酰肌醇4，5-二磷酸(PIP2)途径、蛋白激酶C途径和一条新的途径，我们将其命名为“快速途径”。快速通路的存在是假设的，因为抑制太快而不能被其他通路所解释。我们打算阐明这一途径的机制。我们还打算研究QUICK和PIP2通路之间可能的协同关系。

DESCRIPTION (provided by applicant): Ion channels are the major determinant for excitability changes of neurons. Our long-term objective is to elucidate the mechanism by which slow excitation and slow inhibition of brain neurons take place. These events occur on time scales of tens of seconds to a few minutes, and are mostly mediated by G proteins. Our immediate research will focus on clarifying the signal transduction mechanism by which G-protein-coupled inward rectifier Kv (GIRK) channels are modulated by substance P (SP), an excitatory peptide neurotransmitter. Native cultured noradrenergic neurons in the locus coeruleus (LC) from newborn rats and mice, as well ascloned GIRKs expressed in HEK293 cells will be used. Electrophysiological and molecular biological techniques will be employed for the investigation. The LC contains neurons that innervate and supply norepinephrine to a wide area of the brain, and plays a vital role in arousal and alertness. Furthermore, LC neurons often degenerate in Alzheimer's disease. LC neurons are dually regulated by opposing signals: inhibitory transmitters, such as somatostatin, activate GIRK channels, whereas excitatory transmitters, such as SP, inhibit the GIRK activity that is activated by somatostatin. The mechanism of the GIRK inhibition, however, is controversial and yet to be determined. The goal of the proposed project is to elucidate the signal transduction mechanism of the SP-induced GIRK channel inhibition. Three possible signal transduction pathways could be responsible for the SP-induced GIRK channel inhibition: the phosphatidyl inositol 4,5-bisphosphate (PIP2) pathway, the protein kinase C pathway, and a new pathway, which we designate as the "quick pathway." The existence of the quick pathway is hypothesized because the inhibition is too rapid to be accounted for by the other pathways. We intend to elucidate the mechanism of this pathway. We also intend to investigate the possible synergistic relation between the quick and the PIP2 pathways.