COBRE: UNV MED SCH: P5: REGULATION OF SMOOTH MUSLE TONE BY K+ CHANNELS

COBRE：UNV MED SCH：P5：K 通道平滑肌张力的调节

• 批准号: 7960568
• 负责人: SANG Don KOH
• 金额: $ 19.32万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960568
• 关键词: Actin-Binding Protein; Actins; Activities of Daily Living; Affect; Animals; Bladder; Cells; Centers of Research Excellence; Computer Retrieval of Information on Scientific Projects Database; Cytoskeleton; Experimental Models; Family; Funding; Grant; Hypertrophy; Immunoblotting; Institution; Knockout Mice; Measurement; Mechanics; Mediating; Membrane Potentials; Methods; Monitor; Mus; Nitric Oxide; Obstruction; Performance; Physiological; Potassium Channel; Preparation; Proteomics; Regulation; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Role; Smooth Muscle; Smooth Muscle Myocytes; Source; Stretching; Testing; United States National Institutes of Health; detrusor muscle; member; patch clamp; potassium channel protein TREK-1; pressure; prevent; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In many smooth muscles, stretch activates contraction. However, normal filling of the urinary bladder is accomplished with minimal increase in pressure until the bladder is near functional capacity. Therefore, bladder smooth muscle must stretch and rearrange itself to allow an increase in bladder volume without pressure rise. Although a number of mechanisms are likely to be important in this response, we have recently described stretch-dependent K+ (SDK) channels in colonic smooth muscle that could hyperpolarize membrane potential and prevent activation of contraction. Recently we found SDK channels in urinary bladder. This finding stimulated the following hypotheses: 1) Functional SDK channels are present in the murine bladder and encoded by members of the two-pore family of K+ channels. 2) SDK channels mediate responses to nitric oxide. 3) Hypertrophy of the mouse bladder is accompanied by an increase in the number of functional SDK channels and may affect the performance of the detrusor muscle in these animals. 4) SDK channels are activated and inhibited by interactions with the actin cytoskeleton. In order to test these hypotheses we will use electrophysiological methods including patch-clamp studies of single SDK channels and measurement of membrane potential in intact bladder smooth muscle to investigate the role of the SDK channels in bladder function. In addition, we will use RT-PCR, immunoblot, and immunohistochemical methods to localize TREK-1 channel subunits in bladder. We will develop antisense methods and TREK- 1 knockout mouse to reduce expression or function of SDK channels in smooth muscle myocytes and in intact bladder. We will then characterize the function of these preparations to test hypotheses 1 and 2. In addition, we will implement an experimental model of bladder outlet obstruction and monitor changes in SDK channel expression (RT-PCR, immunoblot, and immunohistochemical methods) and function (electrophysiological and mechanical measurements). We will characterize the interactions between SDK channels and the actin cytoskeleton using pharmacological and antisense methods to disrupt specific interactions and examining the effects on SDK channel function. The specific actin-binding proteins associated with TREK-1 will be identified with assistance from the Cell to Proteomics Interface Core. In conclusion, the characterization of SDK channels channels in bladder will be important to understand the physiological filling mechanisms and the pathological distension.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在许多平滑肌肉中，拉伸会激活收缩。但是，膀胱的正常填充以最小的压力增加，直到膀胱接近功能。因此，膀胱平滑肌必须伸展和重新排列，以使膀胱体积增加而不会增加压力。尽管在这种反应中可能有许多机制很重要，但我们最近描述了结肠平滑肌中的拉伸依赖性K+（SDK）通道，这些通道可能会使膜电位超极化并阻止收缩的激活。 最近，我们在膀胱中发现了SDK频道。这一发现刺激了以下假设：1）鼠膀胱中存在功能性SDK通道，并由K+通道的两孔家族的成员编码。 2）SDK通道介导对一氧化氮的反应。 3）小鼠膀胱的肥大伴随着功能性SDK通道的数量增加，并可能影响这些动物中逼尿肌的性能。 4）与肌动蛋白细胞骨架相互作用激活并抑制SDK通道。为了检验这些假设，我们将使用电生理方法，包括单个SDK通道的贴片钳研究和测量 完整的膀胱平滑肌的膜电位，以研究SDK通道在膀胱功能中的作用。此外，我们将使用RT-PCR，免疫印迹和免疫组织化学方法来定位膀胱中的Trek-1通道亚基。我们将开发反义方法和TREK-1基因敲除小鼠，以减少平滑肌心肌细胞和完整膀胱中SDK通道的表达或功能。然后，我们将表征这些准备工作的功能，以检验假设1和2。此外，我们将实施一个实验模型的膀胱出口障碍物，并监测SDK通道表达（RT-PCR，免疫印迹和免疫组织化学方法）的变化和功能（电生理和机械测量）。我们将使用药理学和反义方法来表征SDK通道与肌动蛋白细胞骨架之间的相互作用，以破坏特定的相互作用并检查对SDK通道功能的影响。与Trek-1相关的特定肌动蛋白结合蛋白将与 从细胞到蛋白质组学界面核心的帮助。 总之，膀胱中SDK通道的表征对于理解生理填充机制和病理扩张非常重要。