Conklin M W, Barone V, Sorrentino V, Coronado R
Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.
Biophys J. 1999 Sep;77(3):1394-403. doi: 10.1016/S0006-3495(99)76988-2.
The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.
在缺乏特定兰尼碱受体（RyR）亚型的基因敲除小鼠中，Ca(2+)火花的动力学行为应能提供有关RyR簇功能和组装的分子信息。我们检测了来自胚胎第18天（E18）小鼠的RyR 3型基因敲除肋间肌管中的静息Ca(2+)火花，并将其与同年龄野生型（wt）小鼠中的Ca(2+)火花以及wt成年小鼠足部快肌细胞中的Ca(2+)火花进行比较。来自RyR 3型基因敲除胚胎细胞的火花（368次事件）明显更小、更短暂，且达到峰值的时间比同年龄wt细胞的火花（280次事件）更快。成年细胞中的火花（220次事件）很少见，但它们具有高度可重复性，群体平均值小于胚胎RyR 3型基因敲除细胞中的平均值，但与成年两栖动物骨骼肌纤维中报道的平均值相似。火花峰值强度（DeltaF/Fo）与半最大强度全宽（FWHM）与半最大强度全持续时间（FTHM）的三维表示表明，wt胚胎火花在大小和动力学方面比成年肌肉中的火花变化大得多。在所有情况下，丁卡因（0.2 mM）消除了Ca(2+)火花活性，而咖啡因（0.1 mM）延长了wt胚胎和成年细胞中火花的持续时间，但在RyR 3型基因敲除细胞中没有。这些结果证实火花源自RyRs。RyR 3型基因敲除细胞对咖啡因的低敏感性与其他研究者的观察结果完全一致。这项研究有三个结论：i）在RyR 3型基因敲除肌管中，在没有其他RyR亚型的情况下，RyR 1型参与Ca(2+)火花活动；ii）在成年哺乳动物骨骼肌细胞中可以检测到参数与成年两栖动物骨骼肌中报道的相似的Ca(2+)火花，尽管频率较低；iii）正常（wt）胚胎肌肉中观察到的异常大的Ca(2+)火花的主要贡献者是RyR 3型。为了解释成年骨骼肌与胚胎骨骼肌相比火花大小的减小，我们认为在胚胎肌肉中，RyR 1型和RyR 3型通道在同一次火花期间共同促进Ca(2+)释放，并且Ca(2+)火花经历了一个成熟过程，其中涉及RyR 3型的减少。
