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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 在许多平滑肌中，拉伸会激活收缩。然而，膀胱的正常充盈是通过最小的压力增加来完成的，直到膀胱接近功能容量。因此，膀胱平滑肌必须伸展并重新排列自身，以允许膀胱容量增加而不增加压力。尽管许多机制可能在这种反应中很重要，但我们最近描述了结肠平滑肌中的拉伸依赖性 K+ (SDK) 通道，它可以使膜电位超极化并防止收缩激活。 最近我们在膀胱中发现了SDK通道。这一发现激发了以下假设：1) 功能性 SDK 通道存在于小鼠膀胱中，并由 K+ 通道双孔家族的成员编码。 2) SDK 通道介导对一氧化氮的反应。 3) 小鼠膀胱肥大伴随着功能性 SDK 通道数量的增加，可能会影响这些动物逼尿肌的性能。 4) SDK 通道通过与肌动蛋白细胞骨架的相互作用而被激活和抑制。为了测试这些假设，我们将使用电生理学方法，包括单个 SDK 通道的膜片钳研究和测量 完整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 敲除小鼠，以减少平滑肌细胞和完整膀胱中 SDK 通道的表达或功能。然后，我们将表征这些制剂的功能，以检验假设 1 和 2。此外，我们将实施膀胱出口梗阻的实验模型，并监测 SDK 通道表达（RT-PCR、免疫印迹和免疫组织化学方法）和功能的变化（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 通道和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被鉴定为 细胞到蛋白质组学接口核心的协助。 总之，膀胱中 SDK 通道的表征对于理解生理充盈机制和病理性扩张非常重要。