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

描述（由申请人提供）：Ca2+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是必不可少的。然而，它们在维持尿失禁的关键器官尿道平滑肌中的功能和潜在机制尚不清楚。本研究旨在了解由肌浆网中2型ryanodine受体（RyR2）的自发开放和它们在尿道平滑肌（USM）质膜中的离子通道靶点引起的高度局部化和短暂的Ca2+瞬态（“Ca2+火花”）。我们的初步研究表明(1)Ca2+火花在USM中只激活ANO1，一个Ca2+激活的Cl- (ClCa)通道，产生自发瞬态内向电流（tics）并使膜去极化，足以开启l型电压依赖性Ca2+通道；(2) RyR2 R176Q突变敲入小鼠（引起人儿茶酚胺能多态性室性心动过速）暴露于咖啡因（一种RyR激动剂）和苯肾上腺素（一种α - 1肾上腺素受体激动剂）时，USM产生的力比正常RyR2小鼠小；(3) ANO1基因缺失导致Ca2+火花无法激活tics，并降低了新生小鼠在咖啡因刺激下的尿道收缩；(4) PE增加了tic，而气体松弛剂一氧化氮（NO）抑制了tic。因此，我们认为USM中的RyR2和ANO1在控制尿道肌原性和神经原性张力中至关重要，它们的功能障碍可能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Ca2+成像，同时进行贴片夹紧、2D和3D蛋白质定位、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（敲入和条件敲除）小鼠。具体来说，我们将利用RyR2 R176Q突变小鼠和正常小鼠，通过控制全局[Ca2+]I、膜电位和尿道张力，确定USM中Ca2+火花作为收缩机制，而不是膀胱和血管平滑肌中的放松机制（Aim 1）。系统性ANO1-/-小鼠非常年轻就死亡，因此很难在成熟小鼠和体内研究ANO1在尿道中的作用。我们获得了一条平滑肌特异性ANO1-/-小鼠系，这些小鼠存活至成熟。通过这个基因敲除系，我们将证实，在成熟小鼠中，ANO1对于维持尿道收缩和压力至关重要，其缺失可能导致尿失禁（目的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RyR2s激活ano1的机制（目的2）。最后，基于我们对PE和NO影响的初步结果，我们将确定PE和NO对RyR2和ANO1的差异调节，分别导致尿道的收缩和松弛（目的3）。我们期望这些研究不仅将显著推进我们对RyR2和ANO1在尿道生理中的作用的理解，而且还将发现新的分子靶点，以开发有效和特异性的治疗尿失禁的方法。

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.