Spectroscopic Probes of Smooth Muscle Functional Dynamics

描述（由申请人提供）：本项目的主要目标是为申请人提供肌肉生物化学和生物物理学方面的严格培训，为他在该领域的独立研究者职业生涯做好准备。他在细胞生理学方面的强大背景将通过肌肉生物物理学的培训得到加强，特别强调收缩肌肉的电子顺磁共振，这是申办者实验室的一个特殊优势。该培训计划的重点是一个研究项目，这是中央申办者的NIH资助的研究。该项目的总体目标是回答有关蛋白质磷酸化和动力学在平滑肌功能中的作用的基本问题，特别强调了解平滑肌的“闩锁”状态，这具有巨大的潜在生物医学重要性。目标：（1）开发一种光谱方法，包括自旋标记和电子顺磁共振（EPR），以真实的时间精确测量皮肤平滑肌纤维中调节轻链（RLC）的磷酸化程度。该方法密切基于申办方实验室的既往出版物。(2)使用这种EPR方法来定量测定等长收缩平滑肌中磷酸化水平、肌力和肌球蛋白分子在弱和强肌动蛋白结合构象状态之间的分布之间的关系。将特别强调肌肉进入闭锁状态的条件，并确定RLC磷酸化对这种状态的影响。(3)第三个目的是探讨无机磷酸盐在扰动收缩过程的机械化学动力学的影响。通过完成这些目标，我们将直接测试平滑肌中闩锁状态的分子机制的关键假设。我们正在测试有关平滑肌调节功能和功能障碍的基本问题。这些问题的答案将提供至关重要的见解，基本的生物化学和生物物理学负责异常平滑肌收缩哮喘和高血压。我们在收缩肌肉中的分子测量可以为药物和其他治疗方法的评估提供基础。

DESCRIPTION (provided by applicant): The primary goal of this project is to provide the applicant with rigorous training in muscle biochemistry and biophysics, to prepare him for a career as an independent investigator in this field. His strong background in cellular physiology will be augmented by training in muscle biophysics, with particular emphasis on electron paramagnetic resonance of contracting muscle, which is a particular strength of the sponsor's laboratory. This training program focuses on a research project that is central to the sponsor's NIH-funded research. The overall goal of this project is to answer fundamental questions about the role of protein phosphorylation and dynamics in the function of smooth muscle, with particular emphasis on understanding the "latch" state of smooth muscle, which has great potential biomedical importance. AIMS: (1) Develop a spectroscopic method, involving spin-labels and electron paramagnetic resonance (EPR), to measure accurately, in real time, the extent of phosphorylation of the regulatory light chain (RLC) in skinned smooth muscle fibers. This method is based closely on previous publications from the sponsor's laboratory. (2) Use this EPR method to determine quantitatively the relationship between phosphorylation level, muscle force, and the distribution of myosin molecules between weak and strong actin binding conformational states, in isometrically contracting smooth muscle. Particular emphasis will be placed on conditions under which the muscle enters the latch state, and to determine the effect of RLC phosphorylation on this state. (3) The third aim probes the effect of inorganic phosphate in perturbing the mechanochemical kinetics of the contraction process. By completing these aims, we will test directly the key hypotheses for the molecular mechanism of the latch state in smooth muscle. PUBLIC HEALTH RELEVANCE We are testing fundamental questions about the function and malfunction of smooth muscle regulation. The answers to these questions will provide crucial insights into the underlying biochemistry and biophysics responsible for abnormal smooth muscle contraction in asthma and hypertension. Our molecular measurements in contracting muscle can provide the basis for evaluation of drugs and other therapeutic approaches.