Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.
Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.
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DOI：
10.1085/jgp.200509475
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发表时间：
2006-06
期刊：
影响因子：
--
通讯作者：
Vergara JL
中科院分区：
文献类型：
--
作者：
Gómez J;Neco P;DiFranco M;Vergara JL
利用自制的双光子激光扫描成像显微镜（TPLSM）和斑点检测功能，研究了小鼠骨骼肌中钙离子释放过程的时空特征。一个双微电极配置用于电刺激肌纤维，记录动作电位（AP），并控制其肌浆组成。我们使用125 μM低亲和力Ca 2+指示剂俄勒冈州绿色488 BAPTA-5 N（OGB-5 N）和5或10 mM Ca 2+螯合剂EGTA（pCa 7），以阻止纤维收缩并限制释放部位附近[Ca 2 +]的变化。图像和斑点数据显示，OGB-5 N荧光沿着纤维的静止分布是均匀的，除了集中在Z线处的窄峰（高于整体荧光的约23%），如它们相对于横小管（T小管）的二-8-ANEPPS染色的非重叠定位所证明的。使用斑点检测，记录从相邻的纵向位置相隔100 nm由AP刺激诱发的局部Ca 2+瞬变。最大和最快的ΔF/F瞬变在Z线两侧的位点检测到，并与T小管共定位;最小和最慢的在M线检测到，而Z线的瞬变显示中间特征。三维重建显示，每个肌节产生两个AP诱发的Ca 2+释放域，它们位于Z线两侧，与T小管共定位。在10 mM细胞内EGTA的存在下，这些结构域在AP的峰值后的1.4 ms内形成，并在1.4 ms内消散。其半峰全宽（FWHM）（在T小管位置的Ca 2+瞬变达到峰值时测量）为0.62 μm，与di-8-ANEPPS曲线测量的0.61 μm相似。这两个值都超过了光学系统的分辨率的限制，但它们的相似性表明，在高[EGTA]的成年哺乳动物肌纤维中的Ca 2+结构域仅限于位于交界肌浆网（SR）的Ca 2+释放位点。
The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca2+ transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest ΔF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca2+ release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in ∼1.4 ms and dissipate within ∼4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca2+ transients peaked at T-tubule locations, was 0.62 μm, similar to the 0.61 μm measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca2+ domains in adult mammalian muscle fibers are confined to Ca2+ release sites located at the junctional sarcoplasmic reticulum (SR).
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DOI：
10.1083/jcb.128.5.893
发表时间：
1995-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Giannini G;Conti A;Mammarella S;Scrobogna M;Sorrentino V
通讯作者：
Sorrentino V
影响因子：
5.5
作者：
DELBONO, O;STEFANI, E
通讯作者：
STEFANI, E
影响因子：
3.4
作者：
CANNELL, MB;ALLEN, DG
通讯作者：
ALLEN, DG
影响因子：
5.5
作者：
Hollingworth, S;Soeller, C;Cannell, MB
通讯作者：
Cannell, MB
影响因子：
5.5
作者：
Kirsch, WG;Uttenweiler, D;Fink, RHA
通讯作者：
Fink, RHA