Philipson K D
Biochim Biophys Acta. 1985 Dec 5;821(2):367-76. doi: 10.1016/0005-2736(85)90107-5.
The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.
心肌肌膜囊泡中的钠钙交换机制可催化肌膜两侧的钙离子与另一侧的钠离子进行交换。关于钠离子和钙离子对细胞内膜表面和细胞外膜表面交换体结合位点的相对亲和力，目前所知甚少。我们之前曾报道过（菲利普森，K.D.和西本，A.Y.（1982年）《生物化学杂志》257卷，5111 - 5117页）一种测量肌膜囊泡混合群体（主要是外翻囊泡）中仅外翻囊泡的钠钙交换的方法。我们得出结论，对于外翻囊泡和内翻囊泡，依赖胞内钠离子的钙离子摄取的表观米氏常数（Km（Ca2 +））相似。在本研究中，我们详细研究了外翻囊泡和囊泡总体的依赖胞外钠离子的钙离子外流情况。为了在测量钙离子外流之前使囊泡加载钙离子，使用了四种方法：1. 钠钙交换；2. 钙离子被动扩散；3. 依赖ATP的钙离子摄取；4. 用通过钠泵主动转运到囊泡中的钠离子交换钙离子。前两种方法使所有肌膜囊泡加载钙离子，而后两种方法选择性地使外翻囊泡加载钙离子。我们能够得出结论，外翻囊泡和内翻囊泡中钙离子外流对胞外钠离子浓度的依赖性相似。因此，钠钙交换体在肌膜的两个表面上的表观米氏常数（Na +）值（约等于30 mM）相似。在其他实验中，胞外钠离子对囊泡总体的依赖胞内钠离子的钙离子摄取的抑制作用比对外翻囊泡的抑制作用更强。显然，钠离子在右向外侧囊泡的外表面上比在外翻囊泡的外表面上更能有效地竞争钙离子结合位点。因此，尽管在不存在另一种离子的情况下对钠离子或钙离子的亲和力似乎是对称的，但在交换体两侧钠离子和钙离子之间的相互作用并不相同。钠钙交换体不是一种完全对称的转运蛋白。
