Function and regulation of elemental Ca2+ signaling in urethral smooth muscle

描述（由申请人提供）：Ca 2+信号和离子通道在控制几种组织类型和器官的平滑肌收缩中至关重要。然而，它们在尿道平滑肌中的功能和潜在机制知之甚少，尿道是维持泌尿系统的关键器官。本提案旨在了解高度本地化和短暂的Ca 2+瞬变（“Ca 2+火花”），导致自发开放的2型兰尼碱受体（RyR 2）在肌浆网，和他们的离子通道靶在质膜在尿道平滑肌（USM）。我们的初步研究表明：（1）USM中的Ca ~（2+）火花仅激活Ca ~（2+）激活的Cl-（ClCa）通道ANO 1，产生自发的瞬时内向电流（STIC），并使膜充分去极化，开启L型电压依赖性Ca ~（2+）通道;（2）来自RyR 2 R176 Q突变的敲入小鼠的USM（其在人类中引起儿茶酚胺能多形性室性心动过速）在暴露于咖啡因（RyR激动剂）和苯肾上腺素（PE）（α 1-肾上腺素能受体激动剂）时产生较小的力，与正常RyR 2小鼠相比;（3）ANO 1的基因缺失导致Ca 2+火花不能激活STIC，并且降低新生小鼠在咖啡因刺激下的尿道收缩;（4）PE增加STIC，并且一氧化氮（NO），一种气体松弛剂，抑制STIC。因此，我们建议，RyR 2和ANO 1在USM是必不可少的管理尿道肌源性和神经源性的张力，其功能障碍可能会导致尿道功能障碍和尿失禁。为了验证这一中心假设，我们将采用一种综合方法，使用高速Ca 2+成像，同时膜片钳，2D和3D蛋白定位，单细胞缩短和组织收缩生物测定，体内尿动力学测试，和转基因（敲入和条件性敲除）小鼠。具体来说，我们将建立在USM钙火花作为一种收缩机制，而不是在膀胱和血管平滑肌的松弛机制，通过控制全球[Ca 2 +]I，膜电位，和尿道张力使用RyR 2 R176 Q突变小鼠和正常小鼠（目的1）。全身性ANO 1-/-小鼠死亡年龄很小，因此很难研究ANO 1在成熟小鼠和体内尿道中的作用。我们已经获得了一系列平滑肌特异性ANO 1-/-小鼠，其活到成熟。利用该敲除系，我们将确定在成熟小鼠中ANO 1对于维持尿道收缩和压力是关键的，并且其缺失可能导致尿失禁（Aim 2）。我们将通过3D成像，通道生物物理学和反应扩散模型进一步揭示RyR 2激活ANO 1的潜在机制（Aim 2）。最后，基于我们对PE和NO影响的初步结果，我们将建立PE和NO差异调节RyR 2和ANO 1，分别导致尿道的收缩和舒张（目的3）。我们希望这些研究不仅能显著提高我们对RyR 2和ANO 1在尿道生理学中作用的理解，还能确定新的分子靶点，用于开发有效和特异性的尿失禁治疗方法。

DESCRIPTION (provided by applicant): Ca2+ signals and ion channels are essential in controlling the contraction of smooth muscle in several tissue types and organs. However, their functions and underlying mechanisms in smooth muscle from urethra, an organ critical for maintaining urinary continence, are poorly understood. The present proposal seeks to understand highly localized and short-lived Ca2+ transients ("Ca2+ sparks") that result from the spontaneous opening of type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum, and their ion channel targets in the plasma membrane in urethral smooth muscle (USM). Our preliminary studies revealed that (1) Ca2+ sparks in USM only activate ANO1, a Ca2+-activated Cl- (ClCa) channel, to produce spontaneous transient inward currents (STICs) and depolarize the membrane sufficiently to turn on L-type voltage-dependent Ca2+ channels; (2) USM from knock-in mice of RyR2 R176Q mutation (which causes catecholaminergic polymorphic ventricular tachycardia in human) generates less force when exposed to caffeine, a RyR agonist, and phenylephrine (PE), an alpha1-adrenergic receptor agonist, than in normal RyR2 mice; (3) genetic deletion of ANO1 causes Ca2+ sparks unable to activate STICs, and decreases the urethral contraction upon stimulation by caffeine in neonatal mice; and (4) PE increases STICs, and nitric oxide (NO), a gas relaxant, inhibits STICs. Thus, we propose that RyR2 and ANO1 in USM are essential for governing urethral myogenic and neurogenic tone, and their malfunction may result in urethral dysfunction and urinary incontinence. To test this central hypothesis we will employ an integrated approach using high-speed Ca2+ imaging with simultaneous patch-clamping, 2D and 3D protein localization, single cell shortening and tissue contraction bioassays, in vivo urodynamics tests, and transgenic (knock-in and conditional knockout) mice. Specifically, we will establish that in USM Ca2+ sparks act as a contractile mechanism, rather than a relaxing mechanism as in bladder and vascular smooth muscle, by controlling global [Ca2+]I, membrane potential, and urethra tone using RyR2 R176Q mutant mice and normal mice (Aim 1). Systemic ANO1-/- mice die very young, so it has been difficult to study the role of ANO1 in urethra in mature mice and in vivo. We have obtained a line of smooth muscle specific ANO1-/- mice which live to maturity. With this knockout line, we will establish that in mature mice ANO1 is critical for the maintenance of urethral contraction and pressure and its deletion likely leads to urinary incontinence (Aim 2). We will further uncover the mechanisms underlying activation of ANO1s by RyR2s with 3D imaging, channel biophysics and reaction-diffusion modeling (Aim 2). Finally, building upon our preliminary results on the effects of PE and NO, we will establish that PE and NO differentially modulate RyR2 and ANO1, resulting in the contraction and relaxation of urethra, respectively (Aim 3). We expect that these studies will not only significantly advance our understanding of the roles of RyR2 and ANO1 in urethral physiology, but also identify novel molecular targets for developing effective and specific treatments for urinary incontinence.