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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在许多平滑肌中，拉伸激活收缩。然而，膀胱的正常充盈是在压力增加最小的情况下完成的，直到膀胱接近功能容量。因此，膀胱平滑肌必须伸展和重新排列自身，以允许膀胱体积增加而不增加压力。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牵张依赖性K+（SDK）通道，其可以提高膜电位并阻止收缩激活。 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通道存在于鼠膀胱中，并且由K+通道的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，并可能影响这些动物逼尿肌的表现。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。为了验证这些假设，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 完整膀胱平滑肌中的膜电位以研究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，以减少平滑肌肌细胞和完整膀胱中SDK通道的表达或功能。然后，我们将描述这些制剂的功能，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（电生理和机械测量）的变化。我们将使用药理学和反义方法来破坏特定的相互作用，并检查对SDK通道功能的影响，来表征SDK通道和肌动蛋白细胞骨架之间的相互作用。与TREK-1相关的特异性肌动蛋白结合蛋白将通过 细胞到蛋白质组学接口核心的帮助。 总之，膀胱中SDK通道的表征对于理解生理充盈机制和病理扩张将是重要的。

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.