Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers.
Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers.
复制标题
DOI：
10.1085/jgp.200709831
复制
发表时间：
2007-12
期刊：
影响因子：
--
通讯作者：
Vergara JL
中科院分区：
文献类型：
--
作者：
DiFranco M;Capote J;Quiñonez M;Vergara JL
采用两种基于荧光共振能量转移（FRET）的混合电压传感系统记录成年小鼠横小管系统（TTS）和骨骼肌纤维表面膜的膜电位变化。法尼基化的EGFP或ECFP（EGFP-F和ECFP-F）用作非移动的FRET供体，并且非荧光（二苦胺[DPA]）或荧光（oxonol染料DiBAC 4（5））亲脂性阴离子用作移动的能量受体。采用体内电穿孔法将pEGFP-F和pECFP-F转染趾短屈肌（FDB）。法尼基化荧光蛋白在TTS和表面膜中有效表达。从荧光蛋白到DPA的共振能量转移产生的电压依赖性光学信号被命名为QRET瞬变，以将其与使用DiBAC 4记录的FRET瞬变区分开来（5）。在表达ECFP-F的纤维中，由动作电位刺激引起的QRET瞬变的峰值ΔF/F是表达EGFP-F的纤维的两倍（7.1% vs. 3.6%）。这些数据提供了一个独特的实验证明的重要性，在FRET的光谱重叠。QRET和FRET信号的电压敏感性被证明对应于电压依赖性的带电受体的移位，这表现为电流记录中的非线性分量。对于DPA，电数据和QRET数据都是通过径向电缆模型模拟预测的，其中电荷移位的最大时间常数为0.6 ms。在DiBAC 4染色的纤维中记录的响应于动作电位的FRET信号显示ΔF/F幅度高达28%，但其上升相比QRET信号慢。模型模拟需要一个时间常数为1.6毫秒的电荷易位，以预测电流和FRET数据。我们的研究结果提供了基础的亲脂性离子作为工具，以测试快速电压依赖性的膜蛋白的构象变化的TTS的潜在用途。
Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC4(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC4(5). The peak ΔF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC4(5) exhibit ΔF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.
登录
查看更多内容
影响因子：
3.4
作者：
DEWEY, TG;HAMMES, GG
通讯作者：
HAMMES, GG
影响因子：
56.9
作者：
GAGE, PW;EISENBERG, RS
通讯作者：
EISENBERG, RS
影响因子：
3.4
作者：
GONZALEZ, JE;TSIEN, RY
通讯作者：
TSIEN, RY
横向管状延迟对哺乳动物骨骼肌电荷运动动力学的影响。
DOI：
10.1085/jgp.85.1.21
发表时间：
1985-01
期刊：
The Journal of general physiology
影响因子：
--
作者：
Simon BJ;Beam KG
通讯作者：
Beam KG
影响因子：
5.5
作者：
ASHCROFT, FM;HEINY, JA;VERGARA, J
通讯作者：
VERGARA, J