Regulation of the mitochondrial Ca2+ uniporter by external adenine nucleotides: the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations.
Regulation of the mitochondrial Ca2+ uniporter by external adenine nucleotides: the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations.
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通过外部腺嘌呤核苷酸调节线粒体 Ca2 单向转运蛋白：单向转运蛋白的行为类似于受核苷酸和二价阳离子调节的门控通道。
DOI：
10.1021/bi970180y
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Pfeiffer,DR
中科院分区：
文献类型：
--
作者：
Litsky,ML;Pfeiffer,DR
我们之前使用解偶联剂强制反向活性的测量来证明线粒体Ca2+单转运蛋白在短暂的快速活性后被外部EGTA和游离Mg2+强烈抑制。使用同样的方法，我们现在表明，除了二价阳离子外，单转运体还受外部腺嘌呤核苷酸和细胞质溶胶的其他组分的调节。精胺（EC0.5≈40 μM）可逆转EGTA +游离Mg2+的抑制作用，并可通过等渗密度梯度法纯化线粒体。在任何一种情况下，外部腺嘌呤核苷酸以浓度依赖的方式恢复抑制。有效顺序为ATP b> ADP > AMP，核苷腺苷无效。在三磷酸核苷酸中，顺序为ATP > CTP≈UTP > GTP。ATP （EC50≈0.6 mM）在线粒体和丝裂体中的有效性与AMPPNP相同，并且不受寡霉素、羧白术苷或AP5A的存在（单独或联合使用）的影响。这些发现表明，ATP通过一种不需要水解的机制作用于内膜外表面的一个位点。在某些条件下（EC50≈20 μM）磷酸也能抑制反向单端口。通过对有丝分裂体进行胰凝乳酶处理，可以区分游离ATP和游离Mg2+抑制单一性转运蛋白的位点。胰凝乳酶可以消除Mg2+的作用，但不影响ATP的作用。数据是在一个模型的背景下解释的，在这个模型中，单转运体被认为是一个门控通道，部分由接受二价阳离子或核苷酸的特定外部效应位点控制。考虑了线粒体对细胞Ca2+调节和基质游离Ca2+浓度对TCA循环活性调节的可能后果。
We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+is reversed by spermine (EC0.5≈ 40 μM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP ≈ UTP > GTP. The effectiveness of ATP (EC50≈ 0.6 mM) is the same in mitochondria and mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxyatractyloside, or AP5A, used alone or in combinations. These findings indicate that ATP acts at a site located on the outer surface of the inner membrane through a mechanism which does not require its hydrolysis. Phosphate also inhibits reverse uniport under some conditions (EC50≈ 20 μM). The sites at which free ATP and free Mg2+inhibit the uniporter can be distinguished by chymotrypsin treatment of mitoplasts, which eliminates the action of Mg2+but does not affect the action of ATP. Data are interpreted within the context of a model in which the uniporter is considered to be a gated channel that is controlled, in part, by specific external effector sites that accept divalent cations or nucleotides. The possible consequences of the model for cell Ca2+regulation by mitochondria and regulation of TCA cycle activity by the matrix free Ca2+concentration are considered.