Murphy Robyn M, Verburg Esther, Lamb Graham D
Department of Zoology, La Trobe University, Bundoora campus, Melbourne, Victoria 3086, Australia.
J Physiol. 2006 Oct 15;576(Pt 2):595-612. doi: 10.1113/jphysiol.2006.114090. Epub 2006 Jul 20.
Skeletal muscle fibres contain ubiquitous and muscle-specific calcium-dependent proteases known as calpains. During normal activity, intracellular [Ca(2+)] in muscle fibres increases to high levels ( approximately 2-20 microm), and it is not apparent how this can be reconciled with the activation properties of the calpains. Calpains evidently do not cause widespread proteolytic damage within muscle fibres under normal circumstances, but do have a role in necrosis in dystrophic muscle fibres. In this study, we examined the in situ localization and regulation of calpains in muscle fibres in order to identify how they are attuned to normal function. The sarcolemma of individual muscle fibres of the rat was removed by microdissection (fibre 'skinning') in order to determine the compartmentalization and diffusibility of the two most Ca(2+)-sensitive calpains, mu-calpain and calpain-3, and to permit precise manipulation of cytoplasmic [Ca(2+)] under physiological in situ conditions. Passive force production in stretched fibres, which indicates the patency of the important elastic structural protein titin, was used as a sensitive assay of the amount of diffusible proteolytic activity in individual fibre segments and in muscle homogenates at set [Ca(2+)]. All calpain-3 is bound tightly within a fibre, whereas most mu-calpain ( approximately 0.2 microm) is initially freely diffusible in the cytoplasm at resting [Ca(2+)] but binds within seconds at high [Ca(2+)]. [Ca(2+)] has to be raised to >/= 2 microm for >/= 1 min to initiate detectable autolysis of mu-calpain and to activate appreciable proteolytic activity. If the [Ca(2+)] is raised sufficiently for long enough to initiate substantial autolysis of mu-calpain, the Ca(2+) sensitivity of the proteolytic activity is greatly increased, and it remains active even at 300 nm Ca(2+), with activity only ceasing if the [Ca(2+)] is decreased to approximately 50 nm Ca(2+), close to the normal resting [Ca(2+)]. These findings on the Ca(2+)- and time-dependent binding, autolytic and proteolytic properties of mu-calpain under physiological conditions demonstrate how it is precisely attuned to avoid uncontrolled proteolytic activity under normal circumstances, and indicate why it could lead to substantial proteolytic damage if resting or localized [Ca(2+)] is elevated, as is likely to occur after eccentric contraction and in dystrophic muscle.
骨骼肌纤维含有被称为钙蛋白酶的普遍存在且肌肉特异性的钙依赖性蛋白酶。在正常活动期间，肌肉纤维中的细胞内[Ca(2+)]会升高到高水平（约2 - 20微摩尔），目前尚不清楚这如何与钙蛋白酶的激活特性相协调。在正常情况下，钙蛋白酶显然不会在肌肉纤维内造成广泛的蛋白水解损伤，但在营养不良的肌肉纤维坏死中确实起作用。在本研究中，我们研究了肌肉纤维中钙蛋白酶的原位定位和调节，以确定它们如何适应正常功能。通过显微解剖（纤维“去皮”）去除大鼠单个肌肉纤维的肌膜，以确定两种对Ca(2+)最敏感的钙蛋白酶，即μ-钙蛋白酶和钙蛋白酶-3的区室化和扩散性，并在生理原位条件下精确操纵细胞质[Ca(2+)]。拉伸纤维中的被动力产生，这表明重要的弹性结构蛋白肌联蛋白的通畅性，被用作在设定的[Ca(2+)]下单个纤维段和肌肉匀浆中可扩散蛋白水解活性量的敏感测定方法。所有的钙蛋白酶-3紧密结合在纤维内，而大多数μ-钙蛋白酶（约0.2微摩尔）在静息[Ca(2+)]时最初在细胞质中可自由扩散，但在高[Ca(2+)]下几秒钟内就会结合。[Ca(2+)]必须升高到≥2微摩尔并持续≥1分钟才能引发可检测到的μ-钙蛋白酶自溶并激活可观的蛋白水解活性。如果[Ca(2+)]升高到足够高并持续足够长的时间以引发μ-钙蛋白酶的大量自溶，蛋白水解活性的Ca(2+)敏感性会大大增加，并且即使在300纳米Ca(2+)时仍保持活性，只有当[Ca(2+)]降低到约50纳米Ca(2+)，接近正常静息[Ca(2+)]时活性才会停止。这些关于μ-钙蛋白酶在生理条件下的Ca(2+)和时间依赖性结合、自溶和蛋白水解特性的发现，证明了它如何在正常情况下精确调节以避免不受控制的蛋白水解活性，并表明如果静息或局部[Ca(2+)]升高，如在离心收缩后和营养不良的肌肉中可能发生的情况，为什么它会导致大量的蛋白水解损伤。
