Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe
Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe
复制标题
DOI：
10.1021/bi981429u
复制
发表时间：
1999-02-02
期刊：
影响因子：
2.9
通讯作者：
Khananshvili, D
中科院分区：
文献类型：
--
作者：
Baazov, D;Wang, XL;Khananshvili, D
作为心肌细胞内主要的钙退出系统，电源性Na+-Ca~(2+)交换在兴奋-收缩偶联过程中暴露于调节因子(如细胞内钙)的快速变化。过去，由于技术限制，交易所对监管因素的动态响应尚未得到解决。在这里，我们描述了使用快速反应的电压敏感染料美蓝-540(M540)监测心肌肌膜囊泡Na+-钙交换电活动的停流方案。Na-O依赖的钙外流的M540信号是通过将含钙囊泡与Na缓冲液混合而产生的，产生160 mm的囊外Na和6µM的无钙。该信号被环肽阻滞剂(FRCRCFa)、钙离子载体(离子霉素)或产生电的解偶联剂(呋喃霉素或FCCP)抑制。钠氧依赖性钙外流的M540信号表现为快速的稳态前爆发(210 S(-1))，然后是缓慢的稳态阶段(小于或等于5 S(-1))。胞外(胞液)镍抑制这两个相，其IC50为0.80+/-0.24 mm。在泡外pH为6.0时，依赖于Na-O的钙外流能够产生M540信号，从而支持低pH下Na+-Ca~(2+)交换的化学计量比没有改变的观点[Khanashvili，D.，et al.(1995年)生物化学34,10290-10297]。当胞外无钙浓度降至0.2mM时，依赖Na-O的钙外流的M540信号消失，这不能用钙缺乏进入胞外(胞液)转运部位来解释，因为依赖Na-O的钙外流的反应是以囊内钙为底物的。这些数据表明，在肌膜小泡中，一个调节的胞内钙位置控制着交换器的活动。这个假定的调控位点的性质与电生理学研究中观察到的“慢”钙调控模式的性质不同。在饱和离子条件下，钠氧依赖的钙外流在直径3000-5000埃的囊泡中产生21 mV/ms的初始速率。假设中心密度为300~400个/亩·m~(-2)，泡孔表面为0.5µm~(-2)，每个泡囊可含有150~200个交换体分子，最大周转率为4000~5600个S(-1)。这一周转上限(无论场地密度是多少)可能会对在生理相关条件下调节细胞内钙进入的交换能力造成相当大的限制。
As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Na-o-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 mu M Ca-free. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Na-o-dependent Ca efflux shows a rapid pre-steady-state burst (210 s(-1)), followed by slow steady-state phase (less than or equal to 5 s(-1)). Extravesicular (cytosolic) Ni inhibits both phases with an IC50 of 0.80 +/- 0.24 mM. At an extravesicular pH of 6.0, the Na-o-dependent Ca efflux is able to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+-Ca2+ exchange is not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry 34, 10290-10297]. The M540 signal of Na-o-dependent Ca efflux is lost when the extravesicular Ca-free concentration drops to 0.2 mu M. This effect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because the reaction of Na-o-dependent Ca efflux utilizes intravesicular Cas a substrate. These data suggest that in sarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of this putative regulatory site do not resemble the properties of the "slow" Ca regulatory mode, observed in electrophysiological studies. Under saturating ionic conditions, the Na-o-dependent Ca efflux generates the initial rates of 21 mV/ms in the vesicles with a diameter of 3000-5000 Angstrom. If a site density of 300-400 exchangers/mu m(2) and a vesicular surface of 0.5 mu m(2) are assumed, each vesicle may contain 150-200 exchanger molecules with a maximal turnover rate of 4000-5600 s(-1). This upper limit for turnover (no matter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Ca entry in the cell under physiologically related conditions.