Time-Resolved Monitoring of Electrogenic Na+-Ca2+ Exchange in the Isolated Cardiac Sarcolemma Vesicles by Using a Rapid-Response Fluorescent Probe
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- 作者：David Baazov ; Xiolan Wang ; and Daniel Khananshvili
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：February 2, 1999
- 年：1999
- 卷：38
- 期：5
- 页码：1435 - 1445
- 全文大小：168K
- 年卷期：v.38,no.5(February 2, 1999)
- ISSN：1520-4995
文摘
As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed torapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. Thedynamic aspects of the exchanger response to regulatory factors have not been resolved in the past dueto technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activityof Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dyeMerocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 
M Cafree. This signal is inhibitedby a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler(valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-stateburst (210 s-1), followed by slow steady-state phase (
5 s-1). Extravesicular (cytosolic) Ni inhibits bothphases with an IC50 of 0.80 ± 0.24 mM. At an extravesicular pH of 6.0, the Nao-dependent Ca efflux isable to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+-Ca2+ exchangeis not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry 34, 10290-10297]. The M540 signalof Nao-dependent Ca efflux is lost when the extravesicular Cafree concentration drops to 0.2 M. Thiseffect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because thereaction of Nao-dependent Ca efflux utilizes intravesicular Ca as a substrate. These data suggest that insarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of thisputative regulatory site do not resemble the properties of the "slow" Ca regulatory mode, observed inelectrophysiological studies. Under saturating ionic conditions, the Nao-dependent Ca efflux generatesthe initial rates of 21 mV/ms in the vesicles with a diameter of 3000-5000 Å. If a site density of 300-400 exchangers/m2 and a vesicular surface of 0.5 m2 are assumed, each vesicle may contain 150-200exchanger molecules with a maximal turnover rate of 4000-5000 s-1. This upper limit for turnover (nomatter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Caentry in the cell under physiologically related conditions.
M Cafree. This signal is inhibitedby a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler(valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-stateburst (210 s-1), followed by slow steady-state phase (5 s-1). Extravesicular (cytosolic) Ni inhibits bothphases with an IC50 of 0.80 ± 0.24 mM. At an extravesicular pH of 6.0, the Nao-dependent Ca efflux isable to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+-Ca2+ exchangeis not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry 34, 10290-10297]. The M540 signalof Nao-dependent Ca efflux is lost when the extravesicular Cafree concentration drops to 0.2 M. Thiseffect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because thereaction of Nao-dependent Ca efflux utilizes intravesicular Ca as a substrate. These data suggest that insarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of thisputative regulatory site do not resemble the properties of the "slow" Ca regulatory mode, observed inelectrophysiological studies. Under saturating ionic conditions, the Nao-dependent Ca efflux generatesthe initial rates of 21 mV/ms in the vesicles with a diameter of 3000-5000 Å. If a site density of 300-400 exchangers/m2 and a vesicular surface of 0.5 m2 are assumed, each vesicle may contain 150-200exchanger molecules with a maximal turnover rate of 4000-5000 s-1. This upper limit for turnover (nomatter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Caentry in the cell under physiologically related conditions.