Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.
Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.
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DOI：
10.1085/jgp.116.1.61
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发表时间：
2000-07-01
期刊：
影响因子：
--
通讯作者：
Berlin JR
中科院分区：
文献类型：
--
作者：
Peluffo RD;Argüello JM;Lingrel JB;Berlin JR
Na,K -ATP 酶在 α 亚基 (Glu779Ala) 的 779 位上含有谷氨酸替换为丙氨酸的氨基酸，在缺乏 K + 的情况下支持高水平的 Na-ATP 酶和电生 Na+–Na+ 交换活性。在 Glu779Ala 酶的微粒体制剂中，Na-ATP 酶活性半最大激活的 Na+ 浓度为 161 ± 14 mM (n = 3)。此外，发现在存在和不存在 20 mM K + 的情况下，800 mM Na+ 的酶活性相似。这些结果表明，Na+ 具有低亲和力，可以像 K+ 一样有效地刺激酶转换。为了进一步了解这种酶活性的机制，在无 K + 溶液中灌注期间，用含有 115 mM Na+ 的贴片电极对表达 Glu779Ala 酶的 HeLa 细胞进行电压钳位。生电 Na+-Na+ 交换被观察为哇巴因可抑制的外向电流，其幅度与细胞外 Na+ (Na+ o) 浓度成正比。在测试的所有 Na+o 浓度 (3–148 mM) 下，交换电流在负膜电位 (V M) 时最大，但随着 V M 变得更正而减小。用 Hill 方程分析每个 V M 处的电流表明，Na+–Na+ 交换具有高亲和力、低容量成分，在 0 mV (K 0 0.5) 时的表观 Na+ o 亲和力为 13.4 ± 0.6 mM，并且具有低亲和力、高容量成分，K 0 0.5 为 120 ± 13 mM (n = 17)。高亲和力和低亲和力交换成分均与 V M 相关，分别消耗膜电介质的 30 ± 3% 和 82 ± 6% (n = 17)。贴片电极溶液中的 2 mM 游离 ADP 会抑制低亲和力交换成分，但不会抑制高亲和力交换成分。这些结果表明，生电Na+-Na+交换的高亲和力成分可以通过Na+o作为低亲和力K+同系物来解释。然而，生电交换的低亲和力成分似乎是由于膜电介质深处的 Na+ 特异性位点上的 Na+o 结合激活的正向酶循环所致。开发了 Na,K -ATP 酶的伪六态模型来模拟这些数据和随附论文的结果（Peluffo, R.D.、J.M. Argüello 和 J.R. Berlin. 2000. J. Gen. Physiol. 116:47–59）。该模型表明，仅细胞外离子依赖性反应动力学的改变就可以解释 Glu779Ala 取代对 Na,K -ATP 酶的影响。
Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the α subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+–Na+ exchange activityin the absence of K +. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 ± 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K +. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K +. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K +-free solutions. Electrogenic Na+–Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+ o) concentration. At all Na+ o concentrations tested (3–148 mM), exchange current was maximal at negative membrane potentials (V M), but decreased as V M became more positive. Analyzing this current at each V M with a Hill equation showed that Na+–Na+ exchange had a high-affinity, low-capacity component with an apparent Na+ o affinity at 0 mV (K 0 0.5) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a K 0 0.5 of 120 ± 13 mM (n = 17). Both high- and low-affinity exchange components were V M dependent, dissipating 30 ± 3% and 82 ± 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+–Na+ exchange could be explained by Na+ o acting as a low-affinity K + congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+ o binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K -ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47–59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K -ATPase.
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DOI：
10.1016/0005-2736(85)90572-3
发表时间：
1985-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
CORNELIUS, F;SKOU, JC
通讯作者：
SKOU, JC
影响因子：
2.4
作者：
HANSEN, UP;GRADMANN, D;SLAYMAN, CL
通讯作者：
SLAYMAN, CL
DOI：
10.1016/0005-2736(70)90251-8
发表时间：
1970-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
LAUGER, P;STARK, G
通讯作者：
STARK, G
影响因子：
56.9
作者：
GADSBY, DC;RAKOWSKI, RF;DEWEER, P
通讯作者：
DEWEER, P
影响因子：
3.8
作者：
DEWEER, P
通讯作者：
DEWEER, P