Department of Physiology, Southwestern Medical Center, Dallas, TX, USA.
Laboratory of Obesity and Aging Research, Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
J Gen Physiol. 2023 Oct 2;155(10). doi: 10.1085/jgp.202213329. Epub 2023 Aug 9.
Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 μm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 μM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.
我们利用光学和电学方法证明，BL6 鼠心肌细胞胞质中的扩散在分子量从 30 增加到 2000 时受到限制>20 倍，这大致与具有 porin 通道尺寸的孔的情况相符。在 25°C 时，Bodipy-FL ATP 的扩散速度比在自由水中慢>40 倍。在所分析的几种荧光团中，结合荧光团的分数范围从 2kD FITC 标记的聚乙二醇的 0.1 到 sulforhodamine 的 0.93。未结合的荧光团以 0.5-8×10-7cm2/s（5-80μm2/s）的速度扩散。对钠/钾泵和藜芦碱修饰的钠通道电流的分析表明，35°C 时钠的扩散几乎不受限制（与尖端平衡的时间常数，约 20s）。使用多种策略，我们估计在 35°C 时，ATP 的扩散速度比在自由水中慢 4 到 8 倍。为了解决限制是由蛋白质还是膜网络引起的问题，我们首先验证了一种蛋白质凝胶，10g%明胶，对扩散的限制具有很强的分子量依赖性。在膜提取的心肌纤维中，通过侧向抽吸限制在大直径的尖端中，溶质扩散的限制程度小于完整的心肌细胞。值得注意的是，从骨骼肌（膈肌）心肌细胞中类似提取的肌纤维的限制较小。用β-七叶皂甙（80μM）透化肌细胞膜的心肌细胞中的溶质扩散与完整心肌细胞中的扩散相似。限制与应变有关，BL6 心肌细胞中的限制是 CD1/J6/129svJ 心肌细胞的两倍。此外，CD1/J6/129svJ 心肌细胞中线粒体 porin VDAC1 缺失的纵向扩散受到两倍的限制，而 WT CD1/J6/129svJ 心肌细胞则没有。因此，线粒体网络限制长距离扩散，同时优化核苷酸在肌丝和线粒体之间的转移。我们预计，肌丝和外线粒体膜施加的扩散限制是总细胞质 AMP 和 ADP（约 10μM）的重要决定因素。然而，扩散向肌丝输送 ATP 的能力仍然是 ATP 消耗的 100 倍。
