紧张性平滑肌自发张力形成的信号调控机制研究

Tonic smooth muscles are able to produce spontaneous tone at constant intracellular calcium without external stimuli, thereby playing essential roles during physiological and pathological processes. Using a knockout animal, we previously found that calcium/calmodulin dependent MLCK and its myosin light chain phosphorylation were required for stimuli-evoked smooth muscle contraction. However, our recent work showed that this calcium-dependent MLCK was also required for spontaneous tone formation where calcium concentration was constant. How MLCK is activated under low intracellular calcium and what is the physiological relevance remains unknown. To address this issue, we intend to systematically investigate the mechanism underlying spontaneous tone formation. We propose that the RyR1-mediated spontaneous calcium spark locally activate MLCK through increased local calcium via depolarization by calcium-activated chloride channel. The local MLCK activity towards myosin light chain phosphrylation may be superposed through a calcium sensitization mechanism. To test this hypothesis, we will use knockout mice and series methods of smooth muscle physiology, and analyze the contractile properties, activation status of the modules converging on signal pathway of MLCK activation and calcium spark production and so on. We will also investigate the relationship of the mechanism we proposed with human diseases. The successful achievement of this project will elucidate a molecular mechanism of spontaneous tone formation and potentially provide a novel clue for the pathogenesis and potential therapeutics of smooth muscle diseases.

紧张性平滑肌细胞在胞内低钙浓度、无刺激时亦能产生自发张力，该张力的形成在血管、呼吸道阻力形成和括约肌功能中发挥着重要作用。我们曾通过基因敲除动物证实：平滑肌的刺激性收缩必须依赖于钙依赖性MLCK及其催化的肌球蛋白磷酸化，最近我们发现，自发张力的形成同样依赖于钙和MLCK，但MLCK是如何在低钙环境中被激活、激活机制是什么、临床意义如何等问题目前还不清楚。为系统研究自发张力形成机制，我们提出：在低钙时，平滑肌可通过RyR1受体介导的钙火花激活钙依赖性氯通道、细胞膜局部去极化和钙内流、进而在局部激活MLCK，由此点燃张力过程。然后由钙敏机制叠加该激活效应，维持自发张力。本课题拟将以系列基因敲除小鼠为基本工具，通过系统的平滑肌生理学手段，分别从不同的角度验证以上机制，同时还探讨该机制与临床疾病的相互关系。本研究的成功，将阐明一个新的平滑肌收缩机制，为平滑肌疾病的病理机制和防治研究提供新依据。

为探讨紧张性平滑肌自发收缩的分子机制，本课题以肛门内括约肌为研究对象，通过系列的基因敲除小鼠、电生理技术以及肌肉生理学技术，成功揭示了肛门内括约肌自发收缩的分子机制。研究发现，肛门内括约肌的收缩乃为Ca2+/CaM/MLCK依赖性收缩，由于平滑肌细胞中有高频钙火花释放、导致TMEM16A激活和氯外流，进而膜去极化激活L-type Cav1.2引起外钙内流，最终激活MLCK引起平滑肌收缩，这一收缩机制可能同样适用于其他平滑肌括约肌。通过该课题的研究，建立了TMEM16A条件性基因敲除小鼠，并利用该小鼠开展了其他领域的系列研究和科研合作，同时也培养了高水平的博士研究生。本项目的相关成果分别发表在国际权威杂志上， 如：Nature Communication、Gastroenterology、J Physiology、J Biological Chemistry等。